Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Svp1p defines a family of phosphatidylinositol 3,5‐bisphosphate effectors

Svp1p defines a family of phosphatidylinositol 3,5‐bisphosphate effectors The EMBO Journal (2004) 23, 1922–1933 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors 1, 2 Stephen K Dove *, Robert C Piper , Introduction 1 3 Robert K McEwen , Jong W Yu , Phosphorylated derivatives of inositol and phosphatidylino- 3 4 Megan C King , David C Hughes , sitol fulfil a striking variety of specific functions in eukaryote 5 5 Jan Thuring , Andrew B Holmes , cells, with their actions executed by effector proteins contain- 6 1 Frank T Cooke , Robert H Michell , ing phosphoinositide-specific binding domains. Diverse pro- 7 3 Peter J Parker and Mark A Lemmon tein modules can serve as phosphoinositide ‘sensors’. These include many PH, FYVE and PX (PhoX) domains (Stenmark School of Biosciences, University of Birmingham, Birmingham, UK, Department of Physiology and Biophysics, University of Iowa, Iowa and Aasland, 1999; Lemmon and Ferguson, 2000; Gillooly City, IA, USA, Department of Biochemistry and Biophysics, University et al, 2001; Ellson et al, 2002). of Pennsylvania School of Medicine, Philadelphia, PA, USA, School of Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P )is Environmental and Applied Sciences, University of Derby, Derby, UK, the most recently identified phosphatidylinositol bispho- Department of Chemistry, University of Cambridge, Cambridge, UK, Department of Biochemistry & Molecular Biology, University College sphate isomer (Dove et al, 1997; Whiteford et al, 1997). All London, London, UK and Protein Phosphorylation Laboratory, Cancer eukaryotes make PtdIns(3,5)P using PtdIns3P 5-kinases Research UK London Research Institute, London, UK related to Saccharomyces cerevisiae Fab1p (Cooke et al, 1998; Gary et al, 1998; Ikonomov et al, 2001). PtdIns(3,5)P Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P ), is essential for membrane recycling from the vacuole/lyso- made by Fab1p, is essential for vesicle recycling from somes (Gary et al, 1998; Ikonomov et al, 2001; Dove et al, vacuole/lysosomal compartments and for protein sorting 2002), for ubiquitin-dependent packaging of proteins into into multivesicular bodies. To isolate PtdIns(3,5)P effec- multivesicular bodies (MVBs) (Odorizzi et al, 1998; Dove tors, we identified Saccharomyces cerevisiae mutants et al, 2002), for growth at high temperature (Yamamoto et al, that display fab1D-like vacuole enlargement, one of which 1995; Cooke et al, 1998; Gary et al, 1998) and for vacuole lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p acidification (Bonangelino et al, 1997). Vac14p and Vac7p are displays PtdIns(3,5)P binding of exquisite specificity, Fab1p regulators (Gary et al, 1998; Bonangelino et al, 2002; GFP-Svp1p localises to the vacuole membrane in a Fab1p- Dove et al, 2002), and PtdIns(3,5)P effector proteins that dependent manner, and svp1D cells fail to recycle a marker may facilitate MVB protein sorting have recently been identi- protein from the vacuole to the Golgi. Cells lacking Svp1p fied (Friant et al, 2003; Whitley et al, 2003). Since deletion of accumulate abnormally large amounts of PtdIns(3,5)P . these effectors does not lead to all the defects associated with These observations identify Svp1p as a PtdIns(3,5)P loss of Fab1p, additional effectors remain to be identified. effector required for PtdIns(3,5)P -dependent membrane Other proteins that can bind PtdIns(3,5)P (Xu et al, 2001; recycling from the vacuole. Other Svp1p-related proteins, Cozier et al, 2002) seem unlikely to mediate any of the known including human and Drosophila homologues, bind effects of this lipid. PtdIns(3,5)P similarly. Svp1p and related proteins almost A single unlobed vacuole that largely fills the cell is certainly fold as b-propellers, and the PtdIns(3,5)P - a hallmark of fab1D yeast that cannot make PtdIns(3,5)P binding site is on the b-propeller. It is likely that many of (Yamamoto et al, 1995) so the loss of proteins that are Fab1p the Svp1p-related proteins that are ubiquitous throughout activators or are needed for the actions of PtdIns(3,5)P the eukaryotes are PtdIns(3,5)P effectors. Svp1p is not should cause a similar phenotype. Using a microscopic screen involved in the contributions of FAB1/PtdIns(3,5)P to starting from this vacuole phenotype, we sought genes whose MVB sorting or to vacuole acidification and so additional disruption phenocopies the fab1D vacuole enlargement PtdIns(3,5)P effectors must exist. and identified the Fab1p regulator Vac14p/Svp2p (Dove The EMBO Journal (2004) 23, 1922–1933. doi:10.1038/ et al, 2002). sj.emboj.7600203; Published online 22 April 2004 Herein we show that Svp1p, another gene identified Subject Categories: membranes & transport in this screen, is a specific PtdIns(3,5)P -binding protein Keywords: ATG18; CVT18; AUT10; lysosome; that participates in the recycling of membrane proteins phosphoinositide from the vacuole to the late endosome. Svp1p is the proto- type member of a new family of phosphoinositide effectors. Results *Corresponding author. Department of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. svp1D cells have a fab1D-like vacuole defect Tel.: þ 44 121 414 8513; Fax: þ 44 121 414 7816; Each Euroscarf strain lacks one non-essential gene. We E-mail: [email protected] screened these for enlarged vacuoles resembling those of fab1D cells, and provisionally termed the identified genes Received: 26 August 2003; accepted: 15 March 2004; published online: 22 April 2004 SVP (Swollen Vacuole Phenotype) (Dove et al, 2002). 1922 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al The YFR021w open reading frame (ORF) encodes SVP1, vacuole defects of svp1D cells. However, overexpressed GFP- which is now termed ATG18 (Klionsky et al, 2003). Figure 1A Svp1p (methionine-free medium) disrupted vacuole function shows DIC images of wild-type, fab1D and svp1D cells, and (Figure 1B). Both observations indicate that Svp1p has a role fluorescence images of the same cells with FM4-64-stained in regulating vacuole morphology. vacuoles. Most fab1D and svp1D cells are markedly enlarged, and both mutants usually have one large vacuole that fills GFP-Svp1p localises to the vacuole membrane much of the cell interior: normal vacuoles are smaller and in a FAB1-dependent manner multilobed. Remarkably, SVP1 is physically separated from Phosphoinositide-binding proteins frequently show inositol FAB1 on chromosome VI by only one ORF (Figure 2A). FAB1 lipid-dependent changes in intracellular localisation. When overexpression does not correct the svp1D vacuole enlarge- GFP-Svp1p was expressed at low levels in wild-type cells, it ment, so suppressed Fab1p expression cannot be its cause localised to the vacuole membrane and to a punctate com- (not shown). partment (Figures 1C and 4B). In contrast, most fab1D cells lacked vacuole-associated GFP-Svp1p (Figure 1C), but the GFP-Svp1p on the punctate compartment remained GFP-Svp1p rescues the vacuole defects of svp1D cells (Figure 1C). Localisation of GFP-Svp1p to the vacuole mem- GFP-Svp1p expression from the repressed MET25 promoter brane, but not to the punctate compartment, is therefore (with 5 mM methionine) corrected the svp1D vacuole enlar- Fab1p-dependent, as would be expected for a PtdIns(3,5)P gement. Rescue was variable and occurred at very low Svp1p effector. expression (Figure 1B). SVP1 disruption therefore causes the Previous work on the SVP1 gene SVP1 has previously been named ATG18, AUT10 and CVT18, reflecting its involvement in AUTophagy, and in Cytoplasm- to-Vacuole protein Targeting (Barth et al, 2001; Guan et al, 2001). None of these studies mentioned vacuole enlargement, possibly because they examined svp1D cells under starvation conditions that tend to provoke vacuole enlargement even in wild-type cells. Svp1p-like proteins are present in all eukaryotes The S. cerevisiae genome encodes two other SVP1-like pro- teins, YPL100w/MAI1 and YGR223c (Georgakopoulos et al, 2001; Barth et al, 2002), which we term HSV1 and HSV2 (Homologous with SVP1), respectively. Their disruption did not cause vacuole enlargement (not shown). GFP-Hsv1p/ Mai1p and GFP-Hsv2p localised to a non-vacuolar punctate compartment and this localisation did not require FAB1 (not shown). Svp1p-like proteins are widespread in all eukaryotes (Figure 2B; Barth et al, 2001). For example, the human and Arabidopsis genomes both encode at least three, and Caenorhabditis elegans and Drosophila melanogaster have two or more (Barth et al, 2001). Gene DKFZp434J154 encodes the most Svp1-like human homologue (labelled hSvp1a in Figures 2B and C). Svp1p and its homologues are multi-WD40 proteins that fold as b-propellers SVP1 encodes a serine-rich 500-amino-acid protein with two previously recognised WD-40 motifs near the centre of the sequence (residues 234–274 and 277–318, within blades 5 and 6 in Figure 2C) (Barth et al, 2001; Guan et al, 2001). The Figure 1 The svp1D phenotype involves vacuole enlargement, and 3D-PSSM fold recognition server (Kelley et al, 2000) predicts GFP-Svp1p localisation at the vacuole membrane is FAB1-depen- dent. (A) Differential interference contrast images and FM4-64 that Svp1p, hSvp1a and Hsv2p fold as seven-bladed b-pro- staining of wild-type, svp1D and fab1D cells, demonstrating the pellers. They show high similarity scores when compared greatly enlarged vacuoles of svp1D cells. (B) Svp1p expression with known seven-bladed b-propellers—the transducin b- corrects the svp1D vacuolar defect (upper images, taken during subunit and the C-terminal domain of Tup1p (Figure 2C). methionine-repressed low-level Svp1p expression), but Svp1p over- expression induces cell vacuolation (lower images, during de- Trypsin cleaves Svp1p at Arg377 (Figures 2B and D, black 1–377 repressed expression following methionine removal). (C) When arrow), yielding Svp1p as a stable fragment (MC King GFP-Svp1p was expressed in svp1D cells, it associated mainly and MA Lemmon, unpublished). Combining the results with the vacuole membrane and large punctate structures. Little from the threading analysis and trypsin cleavage, we suggest or none of the GFP-Svp1p was associated with the vacuole mem- brane in fab1D cells. that the Svp1p b-propeller ends around R377, and that the &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1923 | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 2 Sequence analysis and structural modelling of Svp1p and Svp1p-like proteins. (A) YFR021w/SVP1 is on the right arm of chromosome VI, adjacent to FAB1.(B) ClustalW alignment of some eukaryotic Svp1-like proteins. Light-grey bars identify sequence within the putative b- propeller. The dark-grey bar denotes the B/C insert in blade 4. The black arrow shows the deduced site of trypsin cleavage in Svp1p. The black bar indicates the C-terminal domain outside the b-propeller. (C) Threaded alignment of Svp1p, hSvp1a and Hsv2p with the b-propeller of transducin-b (Sondek et al, 1996). Assignment of blades and b-strands is based on transducin-b. Each sequence was submitted to the 3D-PSSM server (Kelley et al, 2000), and gave a significant (480% certainty) score for alignment with transducin-b. Alignments were slightly adjusted in blades 3, 4 and 7, to maintain consistency within the Svp1p family ClustalW alignments. (D) A linear depiction of the Svp1p domain structure and a cartoon of its probable folded structure. Light ovals represent WD-40 blades, and the black arrow the point of trypsin attack. 1924 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al C-terminal 123 residues form an independent domain (see much bound at saturation, possibly indicating an altered Figure 2D). stoichometry. Sequence alignment (Figure 2C) shows that the predic- GST-Hsv2p and GST-hSvp1a bound to PtdIns(3,5)P ted b-strands of the propeller blades are quite well with high affinity (Figure 3G; 0.5 mM MgCl present) and conserved between Svp1p and its homologues, but the inter- selectivity (not shown): the affinity of GST-Hsv2p was similar vening loops are highly divergent. For instance, most homo- to that of GST-Svp1p. GST-Hsv1p/Mai1p also bound specifi- logues lack the large B/C loop in blade 4 of Svp1p (Figures cally to PtdIns(3,5)P (K B500 nM), as did a GST-Tagg-340- 2 D 2B–D; Barth et al, 2001). The C-terminal domains are also residue Drosophila Svp1p homologue with a very short highly variable. C-terminal tail (CG11975 in Figure 2B; K 200–500 nM) (not shown). It therefore appears that specific PtdIns(3,5)P binding is a Svp1p, Hsv1p, Hsv2p and hSvp1a bind PtdIns(3,5)P 2 2 conserved and functionally important feature of many Svp1p- with high affinity and specificity GFP-Svp1p localises to the vacuole in a FAB1-dependent like proteins. manner, so we determined whether Svp1p might bind PtdIns(3,5)P binds to the b-propeller PtdIns(3,5)P , particularly under ionic conditions like those 2 2 2 þ The PtdIns(3,5)P -binding site of Svp1p does not require in the cytosol (inc. millimolar Mg ). We first used phos- the C-terminal sequence that lies outside the b-propeller: its phoinositide ‘dot blots’ (Kavran et al, 1998) and saw binding of P-labelled GST-Svp1p (Figure 3A) and GST-Hsv2p (not removal did not change PtdIns(3,5)P binding. However, 1–170 171–500 shown) only to PtdIns(3,5)P and PtdIns3P. Svp1p and Svp1p , each of which includes about Since dot-blot assays do not always report a protein’s half of the proposed b-propeller, did not bind PtdIns(3,5)P . native lipid selectivity reliably, we used two more quantita- Some Svp1p homologues have quite large insertions within the b-propeller, for example, between strands 4B and 4C tive approaches to analyse phosphoinositide binding by GST- (Figures 2B–D). An Svp1p construct lacking this loop Svp1p. First, we found that GST-Svp1p bound strongly to D4B/C Affigel beads bearing covalently attached PtdIns(3,5)P (Svp1p ) also bound PtdIns(3,5)P with wild-type speci- 2 2 (Figure 3B, ‘total binding’). Washing with buffer containing ficity and affinity (Figure 4A). PtdIns(3,5)P almost completely displaced the bound GST- It seems that PtdIns(3,5)P binds to the b-propeller. A Svp1p, but other PtdInsP and PtdInsP isomers had no effect multiple alignment highlighted two clusters of basic residues that might be involved. There is a widely conserved basic (Figure 3B). GroPIns(3,5)P , the hydrophilic backbone of sequence at the junction between blades 5 and 6 (QFRRG in PtdIns(3,5)P , did not displace Svp1p (not shown). Svp1p) that includes an invariant RRG, and the basic char- Specific binding of PtdIns(3,5)P to Svp1p therefore in- volves at least two interactions: (a) between the anionic head acter of a sequence at the start of strand 2C (SPRRLR in group and a basic amino-acid cluster; and (b) between Svp1p) is also widely conserved (Figure 2C). FTTG hydrophobic parts of the lipid and a nearby hydrophobic Mutating FRRG to FTTG (Svp1p ) decreased 284 287 284 287 the PtdIns(3,5)P affinity more than 40-fold (Figure 4A). By patch on Svp1p. SPSSLS contrast, changing SPRRLR to SPSSLS (Svp1p ) Surface plasmon resonance (SPR) analysis was undertaken 71 76 71 76 only slightly reduced PtdIns(3,5)P binding (Figure 4A). to obtain a quantitative measure of Svp1p binding to mixed lipid vesicles ‘doped’ with a phosphoinositide (3%, mol/mol) and immobilised on BIACore L1 chips (Yu and Lemmon, Svp1p constructs only correct the svp1D vacuole 2001). GST-Svp1p bound strongly to PtdIns(3,5)P -containing enlargement if they bind PtdIns(3,5)P 2 2 FTTG membranes in the presence of 0.5–2 mM MgCl (apparent GFP-Svp1p expressed in yeast was present in the cytosol and nucleus—none was vacuole-associated (Figure 4B). In K B180 nM; Figure 3G). Since GST-induced dimerisation will SPSSLS potentiate Svp1p binding to phosphoinositides, we also ex- contrast, GFP-Svp1p localised to the vacuole normally amined native Svp1p that was monomeric by gel filtration. (Figure 4B). FTTG This bound PtdIns(3,5)P selectively and with high affinity Expression of Svp1p under control of its own promoter (half-maximal atB500 nM Svp1p) (Figures 3C and D). Svp1p did not correct the vacuole enlargement in svp1D cells (not bound weakly to PtdIns3P, and not to PtdIns(4,5)P , shown), suggesting that Svp1p must bind PtdIns(3,5)P if it 2 2 is to function normally in vacuole membrane trafficking. PtdIns(3,4)P or PtdIns4P. FTTG We compared PtdIns(3,5)P binding by Svp1p with the Unexpectedly, though, overexpressing GFP-Svp1p in interactions between other highly selective phosphoinositide wild-type yeast provoked a vacuole enlargement like that sensor domains and their phosphoinositide ligands caused by wild-type Svp1p (Figure 4C). This suggests that FTTG (Figure 3D). The affinity of the Svp1p/PtdIns(3,5)P interac- GFP-Svp1p remains capable of sequestering something, tion was at least 10-fold greater than between the Hrs1 FYVE probably a soluble protein, that is needed for retrograde membrane trafficking, but cannot deliver it to the domain and PtdIns3P (Sankaran et al, 2001), higher than for PtdIns(3,5)P -rich vacuole. the phospholipase C-d PH domain/PtdIns(4,5)P interaction 1 2 2 (Rebecchi and Pentyala, 2000), and weaker than for PtdIns(3,4)P binding to the DAPP1 PH domain (Ferguson Membrane recycling from the vacuole fails in svp1D et al, 2000) (Figure 3D). cells The PtdIns(3,5)P selectivity of Svp1p was reduced Vacuole enlargement in fab1D, vac7D and vac14/svp2D cells 2 þ when the buffer lacked Mg . Although the PtdIns(3,5) is caused, at least partially, by a failure of membrane recy- P affinity was enhanced (K B18 nM), Svp1p also bound to cling to the late endosome (R Piper, unpublished) (Bryant 2 D PtdIns(3,4)P (K B190 nM) and PtdIns(4,5)P (K B200 nM) et al, 1998). As explained in Figure 5A, this process can be 2 D 2 D (Figures 3E and F), although less avidly and with one-third as detected in vivo by appearance in the Golgi of proteolytically &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1925 | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 3 Svp1p and related proteins bind PtdIns(3,5)P with high affinity and selectivity. (A) A dot-blot assay indicates that GST-Svp1p binds both PtdIns3P and PtdIns(3,5)P .(B) GST-Svp1p bound to PtdIns(3,5)P -derivatised Affigel beads is selectively displaced by exogenous 2 2 PtdIns(3,5)P and not by other PtdinsP isomers or by PtdInsPs. (C) Monomeric Svp1p binds to a DOPC layer ‘doped’ with 3 mol% 2 2 PtdIns(3,5)P but not with other phosphoinositides, as detected by Biacore analysis. (D) The affinity of monomeric Svp1p binding to 2 þ PtdIns(3,5)P -‘doped’ lipids is similar to phosphoinositide affinities in other protein/phosphoinositide combinations. (E, F)Mg at a ‘physiological’ (0.5 mM) concentration is needed for Svp1p to show its full PtdIns(3,5)P selectivity. (G) Comparison of PtdIns(3,5)P binding 2 2 by GST-Svp1p, GST-Hsv2p (S. cerevisiae) and GST-hSvp1a (human). All data are representative of at least three independent experiments. matured RS-ALP (mRS-ALP), a variant of the Pho8p alkaline dependent route. It is processed to mRS-ALP by vacuolar phosphatase that has an FXFXD motif incorporated into its N- Pep4p (stage 2), and the FXFXD motif then directs mRS-ALP terminal region. In stage 1, recently synthesised pro-RS-ALP into the FAB1/PtdIns(3,5)P -dependent retrograde pathway traffics from the Golgi directly to the vacuole, via an AP-3- from the vacuole to late endosomes (stage 3), from whence it 1926 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 5 Svp1p is needed for the recycling of vacuole membrane proteins. (A) Scheme depicting routes of trafficking of RS-ALP trafficking (for explanation, see the text). (B)In svp1D cells, the Golgi contains only pro-RS-ALP. Vph1p serves as a marker for the vacuole membrane, and Vps10p for Golgi membrane (for details, see Materials and methods). that launches membrane into the vacuole-to-late-endosome retrograd trafficking pathway requires the Svp1p/ PtdIns(3,5)P complex. svp1D cells make abnormally large amounts of PtdIns(3,5)P The above observations indicate that some Svp1p functions rely on Fab1p-catalysed PtdIns(3,5)P production, so we checked that svp1D cells can make PtdIns(3,5)P . Lipids were extracted from unstressed H-inositol-labelled cells and from cells that were salt-stressed to provoke rapid PtdIns(3,5)P synthesis (Dove et al, 1997). Figure 4 The b-propeller of Svp1p binds PtdIns(3,5)P .(A) Remarkably, unstressed svp1D cells contained 5–10 times PtdIns(3,5)P binding to GST-Svp1p mutants lacking the blade 4 more PtdIns(3,5)P than wild type: PtdIns4P, PtdIns3P and B/C loop or with a mutated basic patch in b-sheet 2C (SPRRLR to PtdIns(4,5)P levels were normal (Figures 6A and B). There SPSSLS) or b-sheet 5D (FRRG to FTTG). Only the conversion of Arg was four times more PtdIns(3,5)P than PtdIns(4,5)P in salt- residues to Thr in the blade 5 basic patch substantially curtailed 2 2 PtdIns(3,5)P binding. (B) Localisation of the Svp1p mutants in challenged svp1D cells—making PtdIns(3,5)P comprise 6% 2 2 SPSSLS svp1D yeast. Wild type and Svp1p localised similarly, but of the phosphoinositides, more than has been seen in any FTTG Svp1p is no longer on vacuole membranes. The constructs other cell. The fact that vacuole defects persist in svp1D cells were expressed as N-terminal GFP fusions from a single-copy that contain exorbitant amounts of PtdIns(3,5)P underlines pUG36 plasmid under control of the MET25 promoter, with 2 0.3 mM methionine. (C) Despite not associating with the vacuole the fact that svp1D cells do not respond appropriately to FTTG membrane, overexpressed GFP-Svp1p causes vacuolation of PtdIns(3,5)P . 60–70% of wild-type cells (compared with 60–70% of cells when WT Deletion of HSV1 or HSV2, or both, did not change the GFP-Svp1p is overexpressed). The constructs are N-terminal GFP cellular PtdIns(3,5)P complement (not shown). fusions in pUG36, and were grown without methionine for maximal expression. Svp1p is not a PtdIns(3,5)P phosphatase Might PtdIns(3,5)P accumulate in svp1D cells because the goes to the Golgi via the retromer pathway (stage 4). As a missing Svp1p is a PtdIns(3,5)P phosphatase? Although the result, the wild-type Golgi contains both pro-RS-ALP and Svp1p sequence includes no recognisable phosphatase-like mRS-ALP, but no mRS-ALP gets to the Golgi of cells defective motifs, we compared the ability of biologically active GST- in step 3 (e.g. fab1, vac7, vac14 and pep12 mutants). Svp1p to hydrolyse PtdIns(3,5)P with that of the We detected both pro-RS-ALP and mRS-ALP in Golgi PtdIns(3,5)P 3-phosphatase MTMR3 (Walker et al, 2001). membranes from wild-type cells, but the svp1D Golgi con- Under conditions in which GST-MTMR3 rapidly depho- tained no mRS-ALP (Figure 5B). This demonstration of a sphorylated PtdIns(3,5)P , GST-Svp1p showed no activity failure of mRS-ALP recycling, together with the vacuole (not shown). Moreover, wild-type cells that overexpressed enlargement in svp1D cells, suggests that the initial step GFP-Svp1p retained a normal PtdIns(3,5)P complement and &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1927 | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 6 svp1D cells accumulate abnormally large amounts of PtdIns(3,5)P . Anion-exchange HPLC chromatograms of the PtdInsP and PtdInsP complements of wild-type and svp1D cells, and their responses to hyperosmotic stress. The deacylated phosphoinositides eluted in the order: PtdIns3P, PtdIns4P, PtdIns(3,5)P (filled peak) and PtdIns(4,5)P (see lower left panel). The relative amounts of phosphoinositides in 2 2 svp1D cells are also shown in the table. The total phosphoinositide complement is unchanged, but in svp1D cells an abnormally large proportion of this is PtdIns(3,5)P . Cells were labelled to isotopic equilibrium, so relative phosphoinositide concentrations match the relative levels of labelling. The data are representative of at least four experiments. showed a normal increase in PtdIns(3,5)P content following 1997; Abeliovich et al, 2003). However, Pho8D60p matured hyperosmotic stress (not shown). normally in fab1Dcells, so Svp1p does not need to interact with Fab1p and/or PtdIns(3,5)P to fulfil its role in auto- phagy. The autophagic role of Svp1p does not need FAB1 or PtdIns(3,5)P Some FAB1/PtdIns(3,5)P -dependent functions do The observation that svp1D cells fail to initiate autophagy not require Svp1p, Hsv1p or Hsv2p correctly (Barth et al, 2001) led to its previous designation as AUT10. To determine whether Svp1p must bind PtdIns(3,5)P Heat tolerance. fab1D yeast grow poorly and lyse at elevated to play its part in autophagy, we investigated this process temperatures (Yamamoto et al, 1995) (Figure 7B). However, in fab1D cells, which express Svp1p but contain no svp1D cells were almost as heat-tolerant as wild-type cells, PtdIns(3,5)P . and their slight sensitivity at 441C was ameliorated by We assessed autophagy by following the maturation of a simultaneous deletion of HSV1 and HSV2 (data not shown). truncated Pho8p pro-enzyme (Pho8D60p) to active alkaline None of the Svp1p-like proteins is therefore needed for phosphatase. Pho8D60p lacks a transmembrane domain PtdIns(3,5)P to confer heat tolerance to cells. needed for trafficking to the vacuole and is made as an inactive cytosolic protein. Its processing needs intravacuolar Vacuole acidification. Inactivation of VAC14/SVP2, VAC7 or Pep4p, so it only becomes activated when bulk-sequestered FAB1 causes a failure of vacuole acidification, even though cytosol is transferred into the vacuole during starvation- the vacuolar H -ATPase (V-ATPase) localises correctly. It has induced autophagy (Noda et al, 1995; Huang and Klionsky, been suggested that PtdIns(3,5)P might positively regulate 2002; Noda et al, 2002). V-ATPase or somehow stabilise the proton gradient Pho8D60p trafficking was suppressed in svp1D cells (Bonangelino et al, 1997; Gary et al, 2002). (Figure 7A) to the same degree as in cells lacking Apg1p, Quinacrine, which is fluorescent and accumulates in acid- another essential protein (Harding et al, 1996; Matsuura et al, ified intracellular compartments, was used to show the severe 1928 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 7 The role of Svp1p in autophagy is PtdIns(3,5)P -independent, and S. cerevisiae Svp1p-like proteins are not needed for several FAB1/ PtdIns(3,5)P -dependent processes. (A) Autophagy processed Pho8D60p normally in fab1D cells, but not in svp1D cells or in the autophagy mutant apg1D (for details, see Materials and methods). (B) Svp1p-related proteins are not needed to maintain growth at elevated temperatures (for details, see Materials and methods). All strains grew at 231C, so only the 421C plate is shown. Data are representative of those from three or four experiments that gave similar results. (C) Svp1p-related proteins are not needed for vacuole acidification, as assessed by accumulation of the fluorescent weak base quinacrine. (D) Svp1p-related proteins are not needed for the sorting of proteins into MVB, as assessed by the trafficking of GFP-Phm5p. acidification defect of fab1D cells (Gary et al, 1998). When we lysis, faulty vacuole acidification and problems with vacuole stained wild type, fab1D, svp1D and svp1D/hsv1D/hsv2D inheritance (Yamamoto et al, 1995; Odorizzi et al, 1998; Dove triple deletion mutants similarly, vacuole acidification was et al, 2002). Why do fab1D cells display such a disparate set only compromised in the fab1D cells (Figure 7C). Control of of dysfunctions, most or all of which are caused by a lack of acidification must therefore employ a PtdIns(3,5)P -depen- PtdIns(3,5)P ? Do cells have one PtdIns(3,5)P effector pro- 2 2 2 dent pathway that needs none of the known Svp1p-related tein that contributes to multiple cell functions, or does proteins. PtdIns(3,5)P interact with several effectors, with each ful- filling a different function(s)? Protein sorting to the MVB. Cells that lack Fab1p or its With the aim of identifying PtdIns(3,5)P targets, we activator Vac14p do not correctly sort certain proteins into screened the EUROFAN deletion mutant collection for gene MVBs, and irreversible ubiquitination of cargo proteins cor- deletions that cause fab1D-like vacuole enlargement (Dove rects this defect (Odorizzi et al, 1998; Dove et al, 2002). et al, 2002). From this screen, YFR021w/SVP1 was particu- This process can be monitored by assessing traffic to the larly intriguing—it is very near YFR019w/FAB1 on chromo- vacuole of GFP-Phm5 (Dove et al, 2002). This normally goes some VI and all eukaryotes have homologues. into the lumen, but defective sorting mislocates it at the membrane. GFP-Phm5p sorting was normal in svp1D and PtdIns(3,5)P specificity of Svp1p and related proteins triple-deletion svp1D//hsv1D/hsv2D cells, but defective in That GFP-Svp1p localises to the vacuole in a FAB1-dependent fab1Dcells (Figure 7D)—so none of the Svp1p-like proteins manner suggested that Svp1p might be a PtdIns(3,5)P effec- contribute to this PtdIns(3,5)P -dependent process. tor. However, Svp1p and Fab1p also seem to interact directly (Georgakopoulos et al, 2001), so that might also contribute to the vacuole localisation. Others have also concluded that Discussion some Svp1p is localised to a punctate compartment near The first insight into the biology of PtdIns(3,5)P came with the vacuole (Barth et al, 2001). Very gentle lysis disrupted the the discovery that Fab1p is the PtdIns3P 5-kinase that makes vacuole localisation of GFP-Svp1p but not its punctuate this lipid (Cooke et al, 1998; Gary et al, 1998). Cells lacking localisation (Guan et al, 2001), and our evidence also indi- FAB1, or expressing a kinase-inactive version of Fab1p in a cates that different mechanisms regulate Svp1p targeting to fab1D background (F Cooke, unpublished), display a com- each compartment. plex spectrum of dysfunctions, including vacuole enlarge- Under approximately ‘physiological’ conditions of ionic 2 þ ment, mis-sorting of proteins into MVBs, heat-sensitive cell strength and Mg concentration, monomeric Svp1p is a &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1929 | | WD-40 motifs can act as lipid-binding modules SK Dove et al very selective and high-affinity PtdIns(3,5)P -binder, as are two related yeast proteins (Hsv1p and Hsv2p), the Drosophila homologue CG11975 and the human homologue hSvp1a. This suggests that Svp1p and its homologues constitute a substantial family of PtdIns(3,5)P specific effector proteins. These are the first proteins known to bind phosphoinositides through small basic amino-acid patches on a b-propeller structure, and so it is possible that a subset of the many proteins encoding b-propeller/WD-40 motifs may also bind to phosphoinositides. This localisation of the Svp1p PtdIns(3,5)P binding on its b-propeller also suggests a possible structural analogy with the recently discovered binding of a phosphothreonine-containing peptide to the eight-bladed b-propeller of Cdc4p (Orlicky et al, 2003). Cellular roles of Svp1p The normal yeast vacuole comprises an array of large orga- nelles that undergo homotypic fusion with other vacuole elements (Wickner, 2002) and heterotypic fusion with smal- ler organelles, including MVBs (Odorizzi et al, 1998). Following the recruitment of membrane by fusion, the va- cuole and MVB elements may normally re-segregate in a manner similar to that seen in cell-free studies with mamma- lian lysosome/late endosome hybrid organelles (Luzio et al, 2000) or a vesicular intermediate may be involved in vacuole to late endosome trafficking. It is likely that Svp1p is involved in a PtdIns(3,5)P -dependent prebudding step in this vacuole membrane segregation process, as suggested in the model in Figure 8A. When this retrograde trafficking step is blocked, as in fab1D and svp1D cells, far more vacuole membrane Figure 8 The involvement of Fab1p, PtdIns(3,5)P and down- accumulates than in normal cells and the multiple vacuole stream effector proteins in yeast cell functions. (A) Outline of the elements fuse (Yamamoto et al, 1995). cycle of vacuole membrane addition and retrieval for which PtdIns(3,5)P appears to be essential. It is not clear whether retro- Studies of trafficking of the v-SNARE, Vti1p, have con- grade vacuole-to-late-endosome trafficking occurs by re-segregation firmed a defect in this segregation/reformation process when of the vacuole and late endosome or by the traffic of a vesicular PtdIns(3,5)P production is impaired. Vti1p is involved in the intermediate between these structures. (B) A tentative synthesis of fusion of late endosomes/MVBs and the vacuole, and a how the actions of multiple PtdIns(3,5)P effector proteins may contribute to various cell functions (see Discussion). All of these, retrograde pathway normally recycles Vti1p to late endo- except the involvement of Svp1p in autophagy, require the presence somes. This is blocked in vac7D cells (Bryant et al, 1998), in cells of FAB1 and/or PtdIns(3,5)P . which lack the Fab1p activator Vac7p and make little PtdIns(3,5)P (Gary et al, 2002). Second mutations that restore PtdIns(3,5)P to normal levels rescue the vac7D action between Fab1p and Svp1p (Georgakopoulos et al, defects: for example, a suppressive mutation of FIG4, which 2001) suggests a possible mechanism for feedback regulation encodes a phosphoinositide phosphatase (Gary et al, 2002). of PtdIns(3,5)P synthesis. Our RS-ALP trafficking results show that svp1D cells pheno- copy this vac7D defect, almost certainly because Svp1p is the PtdIns(3,5)P effector required for this membrane recycling. Svp1p as one of several PtdIns(3,5)P effectors? 2 2 Our working hypothesis is that the Svp1p/PtdIns(3,5)P Taken with recent reports that Ent3p, Ent5p and Vps24p are complex participates in a specific interaction with some other effectors that contribute to the MVB trafficking functions of protein (or complex), and so directs budding from the PtdIns(3,5)P (Friant et al, 2003; Whitley et al, 2003), our vacuole surface. Folded as a b-propeller, Svp1p is well suited results support the idea that the complex fab1D phenotype is to form a platform for protein–protein interactions, probably caused by malfunctions in several, probably independent, of the peptide-in-groove type (ter Haar et al, 2000). Our PtdIns(3,5)P effector pathways. It is clear that neither Ent3p, autophagy studies make it clear that Svp1p also performs Ent5p nor Vps24p (Friant et al, 2003; Whitley et al, 2003), nor PtdIns(3,5)P -independent vacuole-related functions, and any of the Svp1p-related proteins, participates in the effects of involvement of the same binding partners in these func- PtdIns(3,5)P on vacuole acidification or heat tolerance. tions might constitute a mechanistic link between the Elsewhere, we shall describe Svp3p, an unrelated putative PtdIns(3,5)P -dependent and -independent functions of PtdIns(3,5)P effector that influences PtdIns(3,5)P metabo- 2 2 2 Svp1p (Barth et al, 2001; Guan et al, 2001). lism and appears to contribute to MVB sorting and to heat Since svp1D cells accumulate abnormally large amounts tolerance (SK Dove, unpublished; see Figures 7B and D). of PtdIns(3,5)P , it also seems likely that the Svp1p/ Other observations support the notion that PtdIns(3,5)P 2 2 PtdIns(3,5)P complex either restrains PtdIns(3,5)P synth- exerts its effects through multiple effector pathways. For 2 2 esis or activates PtdIns(3,5)P degradation. The known inter- example, low overexpression of the FAB1 gene in the 1930 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al et al, 1997). Mutants were sequenced and then moved into GST vac14-1 background restores vacuole acidification, but the expression vectors. vacuole remains enlarged. However, vacuole enlargement is only corrected if FAB1 is greatly overexpressed (Bonangelino Expression of GST-Svp1p and GST-Hsv2p et al, 2002; Dove et al, 2002). Similarly, the catalytic site The SVP1 and HSV2 ORFs were cloned into pGEX-6P-1 (Amersham- G2042V/G2045V mutant FAB1 normalises MVB sorting in fab1D Pharmacia) (with a rhinovirus-2C protease cleavage site between GST and the target protein) and pGSTag (Ron and Dressler, 1992). cells but does not correct the vacuole enlargement (Odorizzi Both plasmids were transformed into Escherichia coli BL21 and et al, 1998). expressed as follows: cells were grown in LB þ ampicillin (100 mg/ Figure 8B summarises our current understanding of ml) to an OD of 0.5–0.8, induced with IPTG (200–500 mM, 2–4 h, PtdIns(3,5)P effector pathways. It highlights the fact that 301C), harvested, washed in ice-cold PBS containing protease inhibitors (pepstatin A, 5 mg/ml; leupeptin 5 mg/ml; E-64, 3 mg/ml; none of the currently known PtdIns(3,5)P effectors mediates aprotinin, 5 mg/ml; PMSF, 100 mM), lysed (100 mg/ml lysozyme, PtdIns(3,5)P -dependent vacuole acidification, and therefore 41C, 30 min) and disrupted with glass beads in a BeadBeater suggests that at least one S. cerevisiae PtdIns(3,5)P effector (6  30 s, 41C). Triton X-100 (1%) supernatants from the disrupted still awaits discovery. Furthermore, Hsv1p and Hsv2p are cells (8000 g, 15 min; then 100 000 g, 1 h) were filtered (0.2 mm) and applied to glutathione–sepharose 4B beads (30 min, 41C), which likely to be PtdIns(3,5)P effectors, but their loss causes none were eluted following the manufacturer’s recommendations. of the known FAB1/PtdIns(3,5)P -dependent phenotypes—it Proteins were made 50% in ice-cold glycerol, snap-frozen and is therefore probable that undiscovered PtdIns(3,5)P -depen- stored at 801C. dent cellular processes remain to be found. Phosphoinositide dot blots SVP1 and HSV2 cloned into the pGSTag vector (with a PKA Materials and methods phosphorylation site in the GST linker) were expressed in E. coli 32 32 BL21 (see above) and P-labelled (Kavran et al, 1998). [ P]pro- Most materials were from sources defined previously, and methods teins were filtered (0.2 mm) and used to probe lipid ‘dot blots’ of for the growth of yeast, for FM4-64 vacuolar staining, for the GFP- serial two-fold dilutions of phosphoinositides (from 1000 to Phm5p MVB sorting assay, for quinacrine staining and for lipid 15.6 pmol, in CHCl :CH OH:H 0, 400:56:4, v/v/v) on Hybond C 3 3 2 have been described (Dove et al, 1997, 2002; Dove and Michell, membrane (BioRad). Blots were blocked (3 h, room temperature, 1999; McEwen et al, 1999). The source of all yeast strains used in 10 ml PBS, 0.5 mM MgCl , 5% ECL blocking reagent) and probed 6 32 this study is indicated in Table I. PtdIns4P and PtdIns5P were from with 1–2 10 dpm of [ P]protein (30 min, room temperature). Echelon Inc. (Salt Lake City, UT), and PtdIns(3,4)P , PtdIns(4,5)P , 2 2 Blots were extensively washed (PBS/0.5 mM MgCl ) and radio- PtdIns(3,4,5)P , and PtdIns3P were from Matreya Inc. (Pleasant activity detected by autoradiography. Concentrations of the lipid Gap, PA): all were dipalmitoyl. Dioleoyl-PtdCho (DOPC), dipalmi- stocks were quantified immediately before spotting by wet ashing toyl-PtdSer and PtdIns were from Sigma-Aldrich. Phosphoinositides and phosphate determination (Baginski et al, 1967). and Affigel-linked PtdIns(3,5)P were synthesised as described (Krugmann et al, 2002). For Biacore analysis, PtdIns(3,5)P was Protein binding to Affigel-linked PtdIns(3,5)P from CellSignals Inc. (Lexington, KY). GST-Svp1p (5 mM) was incubated (1 ml PBS containing 0.1% Triton X-100, 10 mM DTT and 50 mM PMSF) with 20ml of Affigel-20 beads Vacuole morphology screen covalently linked to PtdIns(3,5)P , in the presence of micellar This screen was as described (Dove et al, 2002). Subsequent dispersions of various phosphoinositides (10 mM) for 30–60 min at analysis of the identified genes included bioinformatic sifting to 41C. In 10 min or less, the beads were sedimented (12 000 g, 1 min) eliminate genes unrelated to vacuole function and to prioritise and washed in the above buffer (twice) and in 5 mM Hepes/KOH, genes with homologues in many organisms. pH 7.5. The remaining bead-bound protein was solubilised in 40 ml SDS–PAGE sample buffer and resolved on 10% SDS–PAGE minigels 0 0 (made with piperazine diacrylamide (BioRad) rather than N N - Plasmid construction methylene bis-acrylamide, to reduce the silver-staining back- GFP-Svp1p and GFP-Hsv2p were constructed by PCR amplification ground). Gels were fixed and stained (BioRad Silver Stain Plus). of the respective ORFs from yeast genomic DNA using the expand proof-reading DNA polymerase (Roche). The Met-regulated con- struct pUG36-SVP1 was created by ligating the SVP1 ORF cut with Biacore analysis of phosphoinositide binding EcoRI and HindIII into pUG36. pUG36-HSV2 was created by ligating This was carried out exactly as described (Yu and Lemmon, 2001). the complete HSV2 ORF, excised with BamHI and SalI, into pUG36. Positive controls were performed alongside Svp1p experiments, and Transformed yeast were grown to 1 10 cells/ml in SC-Ura-Met and included the PLCd1 PH domain (binds PtdIns(4,5)P ), the DAPP1 visualised as described (Dove et al, 2002). Mutations in the SVP1 PH domain (binds PtdIns(3,4)P ), the FAPP1 PH domain (binds gene were carried out using serial overlap extension PCR (Warrens PtdIns4P) and the Hrs1 FYVE domain (binds PtdIns3P). Lipid Table I Yeast strains used in this study Yeast strain Source Reference BY4742 mat a ura3D0 his3D1 leu2D0 lys2D0 EUROSCARF N/A BY4742 svp1HKANMX4 EUROSCARF N/A BY4742 svp1HKANMX4 hsv1HLEU2 hsv2HHIS3 This study N/A BY4742 hsv1HLEU2 hsv2HHIS3 D Alexandraki Georgakopoulos et al (2001) BY4742 hsv1HKANMX4 EUROSCARF N/A BY4742 hsv2HKANMX4 EUROSCARF N/A BY4742 fab1HKANMX4 EUROSCARF N/A BY4742 fab1HKANMX4 pho8HLEU2 This study N/A BY4742 hsv1HKANMX4 pho8HLEU2 This study N/A BY4742 hsv2HKANMX4 pho8HLEU2 This study N/A BY4742 svp1HKANMX4 pho8HLEU2 This study N/A BY4742 svp2HKANMX4 EUROSCARF N/A BY4742 svp3HKANMX4 EUROSCARF N/A NA: not applicable. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1931 | | WD-40 motifs can act as lipid-binding modules SK Dove et al surfaces were used within 8 h of generation, since signal strength 50 mM Tris–HCl, 5 mM MgCl , 1 mM PMSF and 1 mg/ml pepstatin A, began to decrease within B12 h. pH 9, cooled (15 min) and vortexed with glass beads (8  30 s, with GST was quantitatively removed from GST-Svp1p with PreScis- 30 s on ice between cycles). Lysates were centrifuged (10 000 g, sion protease. When gel-filtered on Superose-6 (Amersham-Phar- 15 min, 41C). The supernatant protein (5 mg) was incubated at 251C macia), the liberated Svp1p eluted as a 60–70 kDa monomer. Svp1p for 3 min in 1 ml of 10 mM p-nitrophenolphosphate, 0.5 M Tris and binding was measured by simultaneously passing it over a 2.5 mM MgCl , pH 8.8, with continuous monitoring of OD . 2 420 phosphoinositide-containing sensor surface and a control DOPC Relative rates of dephosphorylation rates were calculated for the sensor surface, with the control signal subtracted from that from the induced and uninduced cells. phosphoinositide surface. Binding data are plotted as per cent of maximum binding against protein concentration injected. K values Dilution assays for temperature sensitivity of growth were calculated as described (Yu and Lemmon, 2001). Yeast were diluted to 1 10 cells/ml, serially diluted four-fold, and 5 ml samples spotted on replicate plates. One was incubated at 231C Estimating K value and the other at 421C, until wild-type cells formed distinct colonies For Svp1p binding to PtdIns(3,5)P , PtdIns(3,4)P and 2 2 at all dilutions. PtdIns(4,5)P , data were fitted to the following equation: 0 1 ½Prot Assay for retrograde vacuole to late endosome trafficking Cells expressing RS-ALP were spheroplasted as previously de- @ A Percent maximal binding ¼ 100 þ Y ½Prot scribed (Urbanowski and Piper, 2001) and lysed in 300 mM sorbitol, þ 1 20 mM HEPES, pH 7.2, and 1 mM EDTA (HES buffer) containing a where [Prot] is the flowing protein concentration (assumed protease inhibitor cocktail (Complete tm, Boehringer Mannheim). unaffected by binding to the surface), K is the dissociation D Postnuclear supernatants were adjusted to 30% Optiprep and constant and Y corresponds to a residual or background signal. layered beneath 5 ml of HES buffer in an SW41 Beckman Fitting was performed using ORIGIN (MicroCal), with floating K D ultracentrifuge tube. Linear Optiprep gradients were overlaid and Y. (BioComp gradient mixer) and the samples were centrifuged (40 000 rpm, 18 h). Fractions were collected downwards from the [ H]inositol labelling and phosphoinositide analysis top. Proteins were separated by SDS–PAGE and immunoblotted Yeast were labelled in inositol-free media for 5–6 cell divisions, so using monoclonal anti-ALP and anti-Vph1p antibodies (Molecular that changes in [ H] would parallel changes in lipid concentration, Probes) and a polyclonal anti-Vps10p antiserum (Piper et al, 1995). and phosphoinositides were extracted and analysed as described (Cooke et al, 1998). Acknowledgements Assay of Pho8D60p maturation This used a method described by Noda et al (1995). PHO8 was We thank Drs Paul Whitley (University of Bath, UK), Gerald Hammond disrupted in appropriate strains using a pho8HLEU2 cassette, and (UCL, London), Despina Alexandraki (University of Crete), Lois the disruptants were transformed with a plasmid that overexpresses Weisman (Iowa) and Geraint Thomas (UCL, London) for valuable Pho8D60p (pTN3, a gift from Dr T Noda, National Institute for Basic discussions and reagents. This work was funded by the Royal Society Biology, Okazaki, Japan). These strains were grown to (SKD and RHM), the Wellcome Trust (SKD and RHM), the BBSRC (to B1 10 cells/ml, washed and either processed immediately or ABT and JT) and grant 5-ROI-GM56846 from the National Institutes of incubated in a nitrogen-free medium for 8 h at 301C, to induce Health (to MAL). SKD is a Royal Society University Research Fellow. autophagy and then processed. They were suspended in 1 ml of RHM is a Royal Society Research Professor. References Abeliovich H, Zhang C, Dunn Jr WA, Shokat KM, Klionsky DJ for its ability to regulate epidermal growth factor receptor degra- (2003) Chemical genetic analysis of Apg1 reveals a non-kinase dation. J Biol Chem 277: 48730–48736 role in the induction of autophagy. Mol Biol Cell 14: 477–490 Dove SK, Cooke FT, Douglas MR, Sayers LG, Parker PJ, Michell RH Baginski ES, Foa PP, Zak B (1967) Microdetermination of inorganic (1997) Osmotic stress activates phosphatidylinositol-3,5-bispho- phosphate, phospholipids, and total phosphate in biologic mate- sphate synthesis. Nature 390: 187–192 rials. Clin Chem 13: 326–332 Dove SK, McEwen RK, Mayes A, Hughes DC, Beggs JD, Michell RH Barth H, Meiling-Wesse K, Epple UD, Thumm M (2001) Autophagy (2002) Vac14 controls PtdIns(3,5)P synthesis and Fab1-depen- and the cytoplasm to vacuole targeting pathway both require dent protein trafficking to the multivesicular body. Curr Biol 12: Aut10p. FEBS Lett 508: 23–28 885–893 Barth H, Meiling-Wesse K, Epple UD, Thumm M (2002) Mai1p is Dove SK, Michell RH (1999) Analysis of the polyphosphorylated essential for maturation of proaminopeptidase I but not for inositol lipids of S. cerevisiae.In Signalling Research: A Practical autophagy. FEBS Lett 512: 173–179 Approach, Milligan G (ed) pp 255–261. Oxford University Press Bonangelino CJ, Catlett NL, Weisman LS (1997) Vac7p, a novel Ellson CD, Andrews S, Stephens LR, Hawkins PT (2002) The PX vacuolar protein, is required for normal vacuole inheritance and domain: a new phosphoinositide-binding module. J Cell Sci 115: morphology. Mol Cell Biol 17: 6847–6858 1099–1105 Bonangelino CJ, Nau JJ, Duex JE, Brinkman M, Wurmser AE, Ferguson KM, Kavran JM, Sankaran VG, Fournier E, Isakoff SJ, Gary JD, Emr SD, Weisman LS (2002) Osmotic stress-induced Skolnik EY, Lemmon MA (2000) Structural basis for discrimina- increase of phosphatidylinositol 3,5-bisphosphate requires tion of 3-phosphoinositides by pleckstrin homology domains. Mol Vac14p, an activator of the lipid kinase Fab1p. J Cell Biol 156: Cell 6: 373–384 1015–1028 Friant S, Pecheur EI, Eugster A, Michel F, Lefkir Y, Nourrisson Bryant NJ, Piper RC, Weisman LS, Stevens TH (1998) Retrograde D, Letourneur F (2003) Ent3p is a PtdIns(3,5)P effector traffic out of the yeast vacuole to the TGN occurs via the required for protein sorting to the multivesicular body. Dev Cell prevacuolar/endosomal compartment. J Cell Biol 142: 651–663 5: 499–511 Cooke FT, Dove SK, McEwen RK, Painter G, Holmes AB, Hall MN, Gary JD, Sato TK, Stefan CJ, Bonangelino CJ, Weisman LS, Emr SD Michell RH, Parker PJ (1998) The stress-activated phosphatidyli- (2002) Regulation of Fab1 phosphatidylinositol 3-phosphate 5- nositol 3-phosphate 5-kinase Fab1p is essential for vacuole func- kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide tion in S. cerevisiae. Curr Biol 8: 1219–1222 phosphatase family member. Mol Biol Cell 13: 1238–1251 Cozier GE, Carlton J, McGregor AH, Gleeson PA, Teasdale RD, Gary JD, Wurmser AE, Bonangelino CJ, Weisman LS, Emr SD Mellor H, Cullen PJ (2002) The phox homology (PX) domain- (1998) Fab1p is essential for PtdIns(3)P 5-kinase activity and dependent, 3-phosphoinositide-mediated association of sorting the maintenance of vacuolar size and membrane homeostasis. nexin-1 with an early sorting endosomal compartment is required J Cell Biol 143: 65–79 1932 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al Georgakopoulos T, Koutroubas G, Vakonakis I, Tzermia M, Noda T, Suzuki K, Ohsumi Y (2002) Yeast autophagosomes: Prokova V, Voutsina A, Alexandraki D (2001) Functional analysis de novo formation of a membrane structure. Trends Cell Biol 12: of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w 231–235 ORF family suggests relations to mitochondrial/peroxisomal Odorizzi G, Babst M, Emr SD (1998) Fab1p PtdIns(3)P 5-kinase functions and amino acid signalling pathways. Yeast 18: function essential for protein sorting in the multivesicular body. 1155–1171 Cell 95: 847–858 Gillooly DJ, Simonsen A, Stenmark H (2001) Cellular functions of Orlicky S, Tang X, Willems A, Tyers M, Sicheri F (2003) Structural phosphatidylinositol 3-phosphate and FYVE domain proteins. basis for phosphodependent substrate selection and orientation Biochem J 355: 249–258 by the SCFCdc4 ubiquitin ligase. Cell 112: 243–256 Guan J, Stromhaug PE, George MD, Habibzadegah-Tari P, Bevan A, Piper RC, Cooper AA, Yang H, Stevens TH (1995) VPS27 Dunn Jr WA, Klionsky DJ (2001) Cvt18/Gsa12 is required for controls vacuolar and endocytic traffic through a prevacuolar cytoplasm-to-vacuole transport, pexophagy, and autophagy in compartment in Saccharomyces cerevisiae. J Cell Biol 131: Saccharomyces cerevisiae and Pichia pastoris. Mol Biol Cell 12: 603–617 3821–3838 Rebecchi MJ, Pentyala SN (2000) Structure, function, and control Harding TM, Hefner-Gravink A, Thumm M, Klionsky DJ (1996) of phosphoinositide-specific phospholipase C. Physiol Rev 80: Genetic and phenotypic overlap between autophagy and the 1291–1335 cytoplasm to vacuole protein targeting pathway. J Biol Chem Ron D, Dressler H (1992) pGSTag—a versatile bacterial expression 271: 17621–17624 plasmid for enzymatic labeling of recombinant proteins. Huang WP, Klionsky DJ (2002) Autophagy in yeast: a review of the Biotechniques 13: 866–869 molecular machinery. Cell Struct Funct 27: 409–420 Sankaran VG, Klein DE, Sachdeva MM, Lemmon MA (2001) High- Ikonomov OC, Sbrissa D, Shisheva A (2001) Mammalian cell affinity binding of a FYVE domain to phosphatidylinositol 3- morphology and endocytic membrane homeostasis require en- phosphate requires intact phospholipid but not FYVE domain zymatically active phosphoinositide 5-kinase PIKfyve. J Biol oligomerization. Biochemistry 40: 8581–8587 Chem 276: 26141–26147 Sondek J, Bohm A, Lambright DG, Hamm HE, Sigler PB (1996) Kavran JM, Klein DE, Lee A, Falasca M, Isakoff SJ, Skolnik EY, Crystal structure of a G-protein beta gamma dimer at 2.1 A Lemmon MA (1998) Specificity and promiscuity in phosphoinosi- resolution. Nature 379: 369–374 tide binding by pleckstrin homology domains. J Biol Chem 273: Stenmark H, Aasland R (1999) FYVE-finger proteins—effectors of 30497–30508 an inositol lipid. J Cell Sci 112: 4175–4183 Kelley LA, MacCallum RM, Sternberg MJ (2000) Enhanced genome ter Haar E, Harrison SC, Kirchhausen T (2000) Peptide-in-groove annotation using structural profiles in the program 3D-PSSM. interactions link target proteins to the beta-propeller of clathrin. J Mol Biol 299: 499–520 Proc Natl Acad Sci USA 97: 1096–1100 Klionsky DJ, Cregg JM, Dunn Jr WA, Emr SD, Sakai Y, Sandoval IV, Urbanowski JL, Piper RC (2001) Ubiquitin sorts proteins into the Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y intralumenal degradative compartment of the late-endosome/ (2003) A unified nomenclature for yeast autophagy-related vacuole. Traffic 2: 622–630 genes. Dev Cell 5: 539–545 Walker DM, Urbe S, Dove SK, Tenza D, Raposo G, Clague MJ (2001) Krugmann S, Anderson KE, Ridley SH, Risso N, McGregor A, Characterization of MTMR3. An inositol lipid 3-phosphatase with Coadwell J, Davidson K, Eguinoa A, Ellson CD, Lipp P, novel substrate specificity. Curr Biol 11: 1600–1605 Manifava M, Ktistakis N, Painter G, Thuring JW, Cooper MA, Warrens AN, Jones MD, Lechler RI (1997) Splicing by overlap Lim ZY, Holmes AB, Dove SK, Michell RH, Grewal A, Nazarian A, extension by PCR using asymmetric amplification: an improved Erdjument-Bromage H, Tempst P, Stephens LR, Hawkins PT technique for the generation of hybrid proteins of immunological (2002) Identification of ARAP3, a novel PI3K effector regulating interest. Gene 186: 29–35 both Arf and Rho GTPases, by selective capture on phosphoinosi- Whiteford CC, Brearley CA, Ulug ET (1997) Phosphatidylinositol tide affinity matrices. Mol Cell 9: 95–108 3,5-bisphosphate defines a novel PI 3-kinase pathway in resting Lemmon MA, Ferguson KM (2000) Signal-dependent membrane tar- mouse fibroblasts. Biochem J 323: 597–601 geting by pleckstrin homology (PH) domains. Biochem J 350: 1–18 Whitley P, Reaves BJ, Hashimoto M, Riley AM, Potter BV, Luzio JP, Rous BA, Bright NA, Pryor PR, Mullock BM, Piper RC Holman GD (2003) Identification of mammalian Vps24p as an (2000) Lysosome–endosome fusion and lysosome biogenesis. effector of phosphatidylinositol 3,5-bisphosphate-dependent J Cell Sci 113: 1515–1524 endosome compartmentalization. J Biol Chem 278: 38786–38795 Matsuura A, Tsukada M, Wada Y, Ohsumi Y (1997) Apg1p, a novel Wickner W (2002) Yeast vacuoles and membrane fusion pathways. protein kinase required for the autophagic process in EMBO J 21: 1241–1247 Saccharomyces cerevisiae. Gene 192: 245–250 Xu Y, Seet LF, Hanson B, Hong W (2001) The Phox homology (PX) McEwen RK, Dove SK, Cooke FT, Painter GF, Holmes AB, Shisheva domain, a new player in phosphoinositide signalling. Biochem J A, Ohya Y, Parker PJ, Michell RH (1999) Complementation 360: 513–530 analysis in PtdInsP kinase-deficient yeast mutants demonstrates Yamamoto A, DeWald DB, Boronenkov IV, Anderson RA, Emr SD, that Schizosaccharomyces pombe and murine Fab1p homologues Koshland D (1995) Novel PI(4)P 5-kinase homologue, Fab1p, are phosphatidylinositol 3-phosphate 5-kinases. J Biol Chem 274: essential for normal vacuole function and morphology in yeast. 33905–33912 Mol Biol Cell 6: 525–539 Noda T, Matsuura A, Wada Y, Ohsumi Y (1995) Novel system for Yu JW, Lemmon MA (2001) All phox homology (PX) domains from monitoring autophagy in the yeast Saccharomyces cerevisiae. Saccharomyces cerevisiae specifically recognize phosphatidylino- Biochem Biophys Res Commun 210: 126–132 sitol 3-phosphate. J Biol Chem 276: 44179–44184 &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1933 | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Svp1p defines a family of phosphatidylinositol 3,5‐bisphosphate effectors

Download PDF
12 pages

Loading next page...
 
/lp/springer-journals/svp1p-defines-a-family-of-phosphatidylinositol-3-5-bisphosphate-VwRnutp9Le

References (52)

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 2004
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1038/sj.emboj.7600203
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal (2004) 23, 1922–1933 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors 1, 2 Stephen K Dove *, Robert C Piper , Introduction 1 3 Robert K McEwen , Jong W Yu , Phosphorylated derivatives of inositol and phosphatidylino- 3 4 Megan C King , David C Hughes , sitol fulfil a striking variety of specific functions in eukaryote 5 5 Jan Thuring , Andrew B Holmes , cells, with their actions executed by effector proteins contain- 6 1 Frank T Cooke , Robert H Michell , ing phosphoinositide-specific binding domains. Diverse pro- 7 3 Peter J Parker and Mark A Lemmon tein modules can serve as phosphoinositide ‘sensors’. These include many PH, FYVE and PX (PhoX) domains (Stenmark School of Biosciences, University of Birmingham, Birmingham, UK, Department of Physiology and Biophysics, University of Iowa, Iowa and Aasland, 1999; Lemmon and Ferguson, 2000; Gillooly City, IA, USA, Department of Biochemistry and Biophysics, University et al, 2001; Ellson et al, 2002). of Pennsylvania School of Medicine, Philadelphia, PA, USA, School of Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P )is Environmental and Applied Sciences, University of Derby, Derby, UK, the most recently identified phosphatidylinositol bispho- Department of Chemistry, University of Cambridge, Cambridge, UK, Department of Biochemistry & Molecular Biology, University College sphate isomer (Dove et al, 1997; Whiteford et al, 1997). All London, London, UK and Protein Phosphorylation Laboratory, Cancer eukaryotes make PtdIns(3,5)P using PtdIns3P 5-kinases Research UK London Research Institute, London, UK related to Saccharomyces cerevisiae Fab1p (Cooke et al, 1998; Gary et al, 1998; Ikonomov et al, 2001). PtdIns(3,5)P Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P ), is essential for membrane recycling from the vacuole/lyso- made by Fab1p, is essential for vesicle recycling from somes (Gary et al, 1998; Ikonomov et al, 2001; Dove et al, vacuole/lysosomal compartments and for protein sorting 2002), for ubiquitin-dependent packaging of proteins into into multivesicular bodies. To isolate PtdIns(3,5)P effec- multivesicular bodies (MVBs) (Odorizzi et al, 1998; Dove tors, we identified Saccharomyces cerevisiae mutants et al, 2002), for growth at high temperature (Yamamoto et al, that display fab1D-like vacuole enlargement, one of which 1995; Cooke et al, 1998; Gary et al, 1998) and for vacuole lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p acidification (Bonangelino et al, 1997). Vac14p and Vac7p are displays PtdIns(3,5)P binding of exquisite specificity, Fab1p regulators (Gary et al, 1998; Bonangelino et al, 2002; GFP-Svp1p localises to the vacuole membrane in a Fab1p- Dove et al, 2002), and PtdIns(3,5)P effector proteins that dependent manner, and svp1D cells fail to recycle a marker may facilitate MVB protein sorting have recently been identi- protein from the vacuole to the Golgi. Cells lacking Svp1p fied (Friant et al, 2003; Whitley et al, 2003). Since deletion of accumulate abnormally large amounts of PtdIns(3,5)P . these effectors does not lead to all the defects associated with These observations identify Svp1p as a PtdIns(3,5)P loss of Fab1p, additional effectors remain to be identified. effector required for PtdIns(3,5)P -dependent membrane Other proteins that can bind PtdIns(3,5)P (Xu et al, 2001; recycling from the vacuole. Other Svp1p-related proteins, Cozier et al, 2002) seem unlikely to mediate any of the known including human and Drosophila homologues, bind effects of this lipid. PtdIns(3,5)P similarly. Svp1p and related proteins almost A single unlobed vacuole that largely fills the cell is certainly fold as b-propellers, and the PtdIns(3,5)P - a hallmark of fab1D yeast that cannot make PtdIns(3,5)P binding site is on the b-propeller. It is likely that many of (Yamamoto et al, 1995) so the loss of proteins that are Fab1p the Svp1p-related proteins that are ubiquitous throughout activators or are needed for the actions of PtdIns(3,5)P the eukaryotes are PtdIns(3,5)P effectors. Svp1p is not should cause a similar phenotype. Using a microscopic screen involved in the contributions of FAB1/PtdIns(3,5)P to starting from this vacuole phenotype, we sought genes whose MVB sorting or to vacuole acidification and so additional disruption phenocopies the fab1D vacuole enlargement PtdIns(3,5)P effectors must exist. and identified the Fab1p regulator Vac14p/Svp2p (Dove The EMBO Journal (2004) 23, 1922–1933. doi:10.1038/ et al, 2002). sj.emboj.7600203; Published online 22 April 2004 Herein we show that Svp1p, another gene identified Subject Categories: membranes & transport in this screen, is a specific PtdIns(3,5)P -binding protein Keywords: ATG18; CVT18; AUT10; lysosome; that participates in the recycling of membrane proteins phosphoinositide from the vacuole to the late endosome. Svp1p is the proto- type member of a new family of phosphoinositide effectors. Results *Corresponding author. Department of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. svp1D cells have a fab1D-like vacuole defect Tel.: þ 44 121 414 8513; Fax: þ 44 121 414 7816; Each Euroscarf strain lacks one non-essential gene. We E-mail: [email protected] screened these for enlarged vacuoles resembling those of fab1D cells, and provisionally termed the identified genes Received: 26 August 2003; accepted: 15 March 2004; published online: 22 April 2004 SVP (Swollen Vacuole Phenotype) (Dove et al, 2002). 1922 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al The YFR021w open reading frame (ORF) encodes SVP1, vacuole defects of svp1D cells. However, overexpressed GFP- which is now termed ATG18 (Klionsky et al, 2003). Figure 1A Svp1p (methionine-free medium) disrupted vacuole function shows DIC images of wild-type, fab1D and svp1D cells, and (Figure 1B). Both observations indicate that Svp1p has a role fluorescence images of the same cells with FM4-64-stained in regulating vacuole morphology. vacuoles. Most fab1D and svp1D cells are markedly enlarged, and both mutants usually have one large vacuole that fills GFP-Svp1p localises to the vacuole membrane much of the cell interior: normal vacuoles are smaller and in a FAB1-dependent manner multilobed. Remarkably, SVP1 is physically separated from Phosphoinositide-binding proteins frequently show inositol FAB1 on chromosome VI by only one ORF (Figure 2A). FAB1 lipid-dependent changes in intracellular localisation. When overexpression does not correct the svp1D vacuole enlarge- GFP-Svp1p was expressed at low levels in wild-type cells, it ment, so suppressed Fab1p expression cannot be its cause localised to the vacuole membrane and to a punctate com- (not shown). partment (Figures 1C and 4B). In contrast, most fab1D cells lacked vacuole-associated GFP-Svp1p (Figure 1C), but the GFP-Svp1p on the punctate compartment remained GFP-Svp1p rescues the vacuole defects of svp1D cells (Figure 1C). Localisation of GFP-Svp1p to the vacuole mem- GFP-Svp1p expression from the repressed MET25 promoter brane, but not to the punctate compartment, is therefore (with 5 mM methionine) corrected the svp1D vacuole enlar- Fab1p-dependent, as would be expected for a PtdIns(3,5)P gement. Rescue was variable and occurred at very low Svp1p effector. expression (Figure 1B). SVP1 disruption therefore causes the Previous work on the SVP1 gene SVP1 has previously been named ATG18, AUT10 and CVT18, reflecting its involvement in AUTophagy, and in Cytoplasm- to-Vacuole protein Targeting (Barth et al, 2001; Guan et al, 2001). None of these studies mentioned vacuole enlargement, possibly because they examined svp1D cells under starvation conditions that tend to provoke vacuole enlargement even in wild-type cells. Svp1p-like proteins are present in all eukaryotes The S. cerevisiae genome encodes two other SVP1-like pro- teins, YPL100w/MAI1 and YGR223c (Georgakopoulos et al, 2001; Barth et al, 2002), which we term HSV1 and HSV2 (Homologous with SVP1), respectively. Their disruption did not cause vacuole enlargement (not shown). GFP-Hsv1p/ Mai1p and GFP-Hsv2p localised to a non-vacuolar punctate compartment and this localisation did not require FAB1 (not shown). Svp1p-like proteins are widespread in all eukaryotes (Figure 2B; Barth et al, 2001). For example, the human and Arabidopsis genomes both encode at least three, and Caenorhabditis elegans and Drosophila melanogaster have two or more (Barth et al, 2001). Gene DKFZp434J154 encodes the most Svp1-like human homologue (labelled hSvp1a in Figures 2B and C). Svp1p and its homologues are multi-WD40 proteins that fold as b-propellers SVP1 encodes a serine-rich 500-amino-acid protein with two previously recognised WD-40 motifs near the centre of the sequence (residues 234–274 and 277–318, within blades 5 and 6 in Figure 2C) (Barth et al, 2001; Guan et al, 2001). The Figure 1 The svp1D phenotype involves vacuole enlargement, and 3D-PSSM fold recognition server (Kelley et al, 2000) predicts GFP-Svp1p localisation at the vacuole membrane is FAB1-depen- dent. (A) Differential interference contrast images and FM4-64 that Svp1p, hSvp1a and Hsv2p fold as seven-bladed b-pro- staining of wild-type, svp1D and fab1D cells, demonstrating the pellers. They show high similarity scores when compared greatly enlarged vacuoles of svp1D cells. (B) Svp1p expression with known seven-bladed b-propellers—the transducin b- corrects the svp1D vacuolar defect (upper images, taken during subunit and the C-terminal domain of Tup1p (Figure 2C). methionine-repressed low-level Svp1p expression), but Svp1p over- expression induces cell vacuolation (lower images, during de- Trypsin cleaves Svp1p at Arg377 (Figures 2B and D, black 1–377 repressed expression following methionine removal). (C) When arrow), yielding Svp1p as a stable fragment (MC King GFP-Svp1p was expressed in svp1D cells, it associated mainly and MA Lemmon, unpublished). Combining the results with the vacuole membrane and large punctate structures. Little from the threading analysis and trypsin cleavage, we suggest or none of the GFP-Svp1p was associated with the vacuole mem- brane in fab1D cells. that the Svp1p b-propeller ends around R377, and that the &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1923 | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 2 Sequence analysis and structural modelling of Svp1p and Svp1p-like proteins. (A) YFR021w/SVP1 is on the right arm of chromosome VI, adjacent to FAB1.(B) ClustalW alignment of some eukaryotic Svp1-like proteins. Light-grey bars identify sequence within the putative b- propeller. The dark-grey bar denotes the B/C insert in blade 4. The black arrow shows the deduced site of trypsin cleavage in Svp1p. The black bar indicates the C-terminal domain outside the b-propeller. (C) Threaded alignment of Svp1p, hSvp1a and Hsv2p with the b-propeller of transducin-b (Sondek et al, 1996). Assignment of blades and b-strands is based on transducin-b. Each sequence was submitted to the 3D-PSSM server (Kelley et al, 2000), and gave a significant (480% certainty) score for alignment with transducin-b. Alignments were slightly adjusted in blades 3, 4 and 7, to maintain consistency within the Svp1p family ClustalW alignments. (D) A linear depiction of the Svp1p domain structure and a cartoon of its probable folded structure. Light ovals represent WD-40 blades, and the black arrow the point of trypsin attack. 1924 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al C-terminal 123 residues form an independent domain (see much bound at saturation, possibly indicating an altered Figure 2D). stoichometry. Sequence alignment (Figure 2C) shows that the predic- GST-Hsv2p and GST-hSvp1a bound to PtdIns(3,5)P ted b-strands of the propeller blades are quite well with high affinity (Figure 3G; 0.5 mM MgCl present) and conserved between Svp1p and its homologues, but the inter- selectivity (not shown): the affinity of GST-Hsv2p was similar vening loops are highly divergent. For instance, most homo- to that of GST-Svp1p. GST-Hsv1p/Mai1p also bound specifi- logues lack the large B/C loop in blade 4 of Svp1p (Figures cally to PtdIns(3,5)P (K B500 nM), as did a GST-Tagg-340- 2 D 2B–D; Barth et al, 2001). The C-terminal domains are also residue Drosophila Svp1p homologue with a very short highly variable. C-terminal tail (CG11975 in Figure 2B; K 200–500 nM) (not shown). It therefore appears that specific PtdIns(3,5)P binding is a Svp1p, Hsv1p, Hsv2p and hSvp1a bind PtdIns(3,5)P 2 2 conserved and functionally important feature of many Svp1p- with high affinity and specificity GFP-Svp1p localises to the vacuole in a FAB1-dependent like proteins. manner, so we determined whether Svp1p might bind PtdIns(3,5)P binds to the b-propeller PtdIns(3,5)P , particularly under ionic conditions like those 2 2 2 þ The PtdIns(3,5)P -binding site of Svp1p does not require in the cytosol (inc. millimolar Mg ). We first used phos- the C-terminal sequence that lies outside the b-propeller: its phoinositide ‘dot blots’ (Kavran et al, 1998) and saw binding of P-labelled GST-Svp1p (Figure 3A) and GST-Hsv2p (not removal did not change PtdIns(3,5)P binding. However, 1–170 171–500 shown) only to PtdIns(3,5)P and PtdIns3P. Svp1p and Svp1p , each of which includes about Since dot-blot assays do not always report a protein’s half of the proposed b-propeller, did not bind PtdIns(3,5)P . native lipid selectivity reliably, we used two more quantita- Some Svp1p homologues have quite large insertions within the b-propeller, for example, between strands 4B and 4C tive approaches to analyse phosphoinositide binding by GST- (Figures 2B–D). An Svp1p construct lacking this loop Svp1p. First, we found that GST-Svp1p bound strongly to D4B/C Affigel beads bearing covalently attached PtdIns(3,5)P (Svp1p ) also bound PtdIns(3,5)P with wild-type speci- 2 2 (Figure 3B, ‘total binding’). Washing with buffer containing ficity and affinity (Figure 4A). PtdIns(3,5)P almost completely displaced the bound GST- It seems that PtdIns(3,5)P binds to the b-propeller. A Svp1p, but other PtdInsP and PtdInsP isomers had no effect multiple alignment highlighted two clusters of basic residues that might be involved. There is a widely conserved basic (Figure 3B). GroPIns(3,5)P , the hydrophilic backbone of sequence at the junction between blades 5 and 6 (QFRRG in PtdIns(3,5)P , did not displace Svp1p (not shown). Svp1p) that includes an invariant RRG, and the basic char- Specific binding of PtdIns(3,5)P to Svp1p therefore in- volves at least two interactions: (a) between the anionic head acter of a sequence at the start of strand 2C (SPRRLR in group and a basic amino-acid cluster; and (b) between Svp1p) is also widely conserved (Figure 2C). FTTG hydrophobic parts of the lipid and a nearby hydrophobic Mutating FRRG to FTTG (Svp1p ) decreased 284 287 284 287 the PtdIns(3,5)P affinity more than 40-fold (Figure 4A). By patch on Svp1p. SPSSLS contrast, changing SPRRLR to SPSSLS (Svp1p ) Surface plasmon resonance (SPR) analysis was undertaken 71 76 71 76 only slightly reduced PtdIns(3,5)P binding (Figure 4A). to obtain a quantitative measure of Svp1p binding to mixed lipid vesicles ‘doped’ with a phosphoinositide (3%, mol/mol) and immobilised on BIACore L1 chips (Yu and Lemmon, Svp1p constructs only correct the svp1D vacuole 2001). GST-Svp1p bound strongly to PtdIns(3,5)P -containing enlargement if they bind PtdIns(3,5)P 2 2 FTTG membranes in the presence of 0.5–2 mM MgCl (apparent GFP-Svp1p expressed in yeast was present in the cytosol and nucleus—none was vacuole-associated (Figure 4B). In K B180 nM; Figure 3G). Since GST-induced dimerisation will SPSSLS potentiate Svp1p binding to phosphoinositides, we also ex- contrast, GFP-Svp1p localised to the vacuole normally amined native Svp1p that was monomeric by gel filtration. (Figure 4B). FTTG This bound PtdIns(3,5)P selectively and with high affinity Expression of Svp1p under control of its own promoter (half-maximal atB500 nM Svp1p) (Figures 3C and D). Svp1p did not correct the vacuole enlargement in svp1D cells (not bound weakly to PtdIns3P, and not to PtdIns(4,5)P , shown), suggesting that Svp1p must bind PtdIns(3,5)P if it 2 2 is to function normally in vacuole membrane trafficking. PtdIns(3,4)P or PtdIns4P. FTTG We compared PtdIns(3,5)P binding by Svp1p with the Unexpectedly, though, overexpressing GFP-Svp1p in interactions between other highly selective phosphoinositide wild-type yeast provoked a vacuole enlargement like that sensor domains and their phosphoinositide ligands caused by wild-type Svp1p (Figure 4C). This suggests that FTTG (Figure 3D). The affinity of the Svp1p/PtdIns(3,5)P interac- GFP-Svp1p remains capable of sequestering something, tion was at least 10-fold greater than between the Hrs1 FYVE probably a soluble protein, that is needed for retrograde membrane trafficking, but cannot deliver it to the domain and PtdIns3P (Sankaran et al, 2001), higher than for PtdIns(3,5)P -rich vacuole. the phospholipase C-d PH domain/PtdIns(4,5)P interaction 1 2 2 (Rebecchi and Pentyala, 2000), and weaker than for PtdIns(3,4)P binding to the DAPP1 PH domain (Ferguson Membrane recycling from the vacuole fails in svp1D et al, 2000) (Figure 3D). cells The PtdIns(3,5)P selectivity of Svp1p was reduced Vacuole enlargement in fab1D, vac7D and vac14/svp2D cells 2 þ when the buffer lacked Mg . Although the PtdIns(3,5) is caused, at least partially, by a failure of membrane recy- P affinity was enhanced (K B18 nM), Svp1p also bound to cling to the late endosome (R Piper, unpublished) (Bryant 2 D PtdIns(3,4)P (K B190 nM) and PtdIns(4,5)P (K B200 nM) et al, 1998). As explained in Figure 5A, this process can be 2 D 2 D (Figures 3E and F), although less avidly and with one-third as detected in vivo by appearance in the Golgi of proteolytically &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1925 | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 3 Svp1p and related proteins bind PtdIns(3,5)P with high affinity and selectivity. (A) A dot-blot assay indicates that GST-Svp1p binds both PtdIns3P and PtdIns(3,5)P .(B) GST-Svp1p bound to PtdIns(3,5)P -derivatised Affigel beads is selectively displaced by exogenous 2 2 PtdIns(3,5)P and not by other PtdinsP isomers or by PtdInsPs. (C) Monomeric Svp1p binds to a DOPC layer ‘doped’ with 3 mol% 2 2 PtdIns(3,5)P but not with other phosphoinositides, as detected by Biacore analysis. (D) The affinity of monomeric Svp1p binding to 2 þ PtdIns(3,5)P -‘doped’ lipids is similar to phosphoinositide affinities in other protein/phosphoinositide combinations. (E, F)Mg at a ‘physiological’ (0.5 mM) concentration is needed for Svp1p to show its full PtdIns(3,5)P selectivity. (G) Comparison of PtdIns(3,5)P binding 2 2 by GST-Svp1p, GST-Hsv2p (S. cerevisiae) and GST-hSvp1a (human). All data are representative of at least three independent experiments. matured RS-ALP (mRS-ALP), a variant of the Pho8p alkaline dependent route. It is processed to mRS-ALP by vacuolar phosphatase that has an FXFXD motif incorporated into its N- Pep4p (stage 2), and the FXFXD motif then directs mRS-ALP terminal region. In stage 1, recently synthesised pro-RS-ALP into the FAB1/PtdIns(3,5)P -dependent retrograde pathway traffics from the Golgi directly to the vacuole, via an AP-3- from the vacuole to late endosomes (stage 3), from whence it 1926 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 5 Svp1p is needed for the recycling of vacuole membrane proteins. (A) Scheme depicting routes of trafficking of RS-ALP trafficking (for explanation, see the text). (B)In svp1D cells, the Golgi contains only pro-RS-ALP. Vph1p serves as a marker for the vacuole membrane, and Vps10p for Golgi membrane (for details, see Materials and methods). that launches membrane into the vacuole-to-late-endosome retrograd trafficking pathway requires the Svp1p/ PtdIns(3,5)P complex. svp1D cells make abnormally large amounts of PtdIns(3,5)P The above observations indicate that some Svp1p functions rely on Fab1p-catalysed PtdIns(3,5)P production, so we checked that svp1D cells can make PtdIns(3,5)P . Lipids were extracted from unstressed H-inositol-labelled cells and from cells that were salt-stressed to provoke rapid PtdIns(3,5)P synthesis (Dove et al, 1997). Figure 4 The b-propeller of Svp1p binds PtdIns(3,5)P .(A) Remarkably, unstressed svp1D cells contained 5–10 times PtdIns(3,5)P binding to GST-Svp1p mutants lacking the blade 4 more PtdIns(3,5)P than wild type: PtdIns4P, PtdIns3P and B/C loop or with a mutated basic patch in b-sheet 2C (SPRRLR to PtdIns(4,5)P levels were normal (Figures 6A and B). There SPSSLS) or b-sheet 5D (FRRG to FTTG). Only the conversion of Arg was four times more PtdIns(3,5)P than PtdIns(4,5)P in salt- residues to Thr in the blade 5 basic patch substantially curtailed 2 2 PtdIns(3,5)P binding. (B) Localisation of the Svp1p mutants in challenged svp1D cells—making PtdIns(3,5)P comprise 6% 2 2 SPSSLS svp1D yeast. Wild type and Svp1p localised similarly, but of the phosphoinositides, more than has been seen in any FTTG Svp1p is no longer on vacuole membranes. The constructs other cell. The fact that vacuole defects persist in svp1D cells were expressed as N-terminal GFP fusions from a single-copy that contain exorbitant amounts of PtdIns(3,5)P underlines pUG36 plasmid under control of the MET25 promoter, with 2 0.3 mM methionine. (C) Despite not associating with the vacuole the fact that svp1D cells do not respond appropriately to FTTG membrane, overexpressed GFP-Svp1p causes vacuolation of PtdIns(3,5)P . 60–70% of wild-type cells (compared with 60–70% of cells when WT Deletion of HSV1 or HSV2, or both, did not change the GFP-Svp1p is overexpressed). The constructs are N-terminal GFP cellular PtdIns(3,5)P complement (not shown). fusions in pUG36, and were grown without methionine for maximal expression. Svp1p is not a PtdIns(3,5)P phosphatase Might PtdIns(3,5)P accumulate in svp1D cells because the goes to the Golgi via the retromer pathway (stage 4). As a missing Svp1p is a PtdIns(3,5)P phosphatase? Although the result, the wild-type Golgi contains both pro-RS-ALP and Svp1p sequence includes no recognisable phosphatase-like mRS-ALP, but no mRS-ALP gets to the Golgi of cells defective motifs, we compared the ability of biologically active GST- in step 3 (e.g. fab1, vac7, vac14 and pep12 mutants). Svp1p to hydrolyse PtdIns(3,5)P with that of the We detected both pro-RS-ALP and mRS-ALP in Golgi PtdIns(3,5)P 3-phosphatase MTMR3 (Walker et al, 2001). membranes from wild-type cells, but the svp1D Golgi con- Under conditions in which GST-MTMR3 rapidly depho- tained no mRS-ALP (Figure 5B). This demonstration of a sphorylated PtdIns(3,5)P , GST-Svp1p showed no activity failure of mRS-ALP recycling, together with the vacuole (not shown). Moreover, wild-type cells that overexpressed enlargement in svp1D cells, suggests that the initial step GFP-Svp1p retained a normal PtdIns(3,5)P complement and &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1927 | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 6 svp1D cells accumulate abnormally large amounts of PtdIns(3,5)P . Anion-exchange HPLC chromatograms of the PtdInsP and PtdInsP complements of wild-type and svp1D cells, and their responses to hyperosmotic stress. The deacylated phosphoinositides eluted in the order: PtdIns3P, PtdIns4P, PtdIns(3,5)P (filled peak) and PtdIns(4,5)P (see lower left panel). The relative amounts of phosphoinositides in 2 2 svp1D cells are also shown in the table. The total phosphoinositide complement is unchanged, but in svp1D cells an abnormally large proportion of this is PtdIns(3,5)P . Cells were labelled to isotopic equilibrium, so relative phosphoinositide concentrations match the relative levels of labelling. The data are representative of at least four experiments. showed a normal increase in PtdIns(3,5)P content following 1997; Abeliovich et al, 2003). However, Pho8D60p matured hyperosmotic stress (not shown). normally in fab1Dcells, so Svp1p does not need to interact with Fab1p and/or PtdIns(3,5)P to fulfil its role in auto- phagy. The autophagic role of Svp1p does not need FAB1 or PtdIns(3,5)P Some FAB1/PtdIns(3,5)P -dependent functions do The observation that svp1D cells fail to initiate autophagy not require Svp1p, Hsv1p or Hsv2p correctly (Barth et al, 2001) led to its previous designation as AUT10. To determine whether Svp1p must bind PtdIns(3,5)P Heat tolerance. fab1D yeast grow poorly and lyse at elevated to play its part in autophagy, we investigated this process temperatures (Yamamoto et al, 1995) (Figure 7B). However, in fab1D cells, which express Svp1p but contain no svp1D cells were almost as heat-tolerant as wild-type cells, PtdIns(3,5)P . and their slight sensitivity at 441C was ameliorated by We assessed autophagy by following the maturation of a simultaneous deletion of HSV1 and HSV2 (data not shown). truncated Pho8p pro-enzyme (Pho8D60p) to active alkaline None of the Svp1p-like proteins is therefore needed for phosphatase. Pho8D60p lacks a transmembrane domain PtdIns(3,5)P to confer heat tolerance to cells. needed for trafficking to the vacuole and is made as an inactive cytosolic protein. Its processing needs intravacuolar Vacuole acidification. Inactivation of VAC14/SVP2, VAC7 or Pep4p, so it only becomes activated when bulk-sequestered FAB1 causes a failure of vacuole acidification, even though cytosol is transferred into the vacuole during starvation- the vacuolar H -ATPase (V-ATPase) localises correctly. It has induced autophagy (Noda et al, 1995; Huang and Klionsky, been suggested that PtdIns(3,5)P might positively regulate 2002; Noda et al, 2002). V-ATPase or somehow stabilise the proton gradient Pho8D60p trafficking was suppressed in svp1D cells (Bonangelino et al, 1997; Gary et al, 2002). (Figure 7A) to the same degree as in cells lacking Apg1p, Quinacrine, which is fluorescent and accumulates in acid- another essential protein (Harding et al, 1996; Matsuura et al, ified intracellular compartments, was used to show the severe 1928 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al Figure 7 The role of Svp1p in autophagy is PtdIns(3,5)P -independent, and S. cerevisiae Svp1p-like proteins are not needed for several FAB1/ PtdIns(3,5)P -dependent processes. (A) Autophagy processed Pho8D60p normally in fab1D cells, but not in svp1D cells or in the autophagy mutant apg1D (for details, see Materials and methods). (B) Svp1p-related proteins are not needed to maintain growth at elevated temperatures (for details, see Materials and methods). All strains grew at 231C, so only the 421C plate is shown. Data are representative of those from three or four experiments that gave similar results. (C) Svp1p-related proteins are not needed for vacuole acidification, as assessed by accumulation of the fluorescent weak base quinacrine. (D) Svp1p-related proteins are not needed for the sorting of proteins into MVB, as assessed by the trafficking of GFP-Phm5p. acidification defect of fab1D cells (Gary et al, 1998). When we lysis, faulty vacuole acidification and problems with vacuole stained wild type, fab1D, svp1D and svp1D/hsv1D/hsv2D inheritance (Yamamoto et al, 1995; Odorizzi et al, 1998; Dove triple deletion mutants similarly, vacuole acidification was et al, 2002). Why do fab1D cells display such a disparate set only compromised in the fab1D cells (Figure 7C). Control of of dysfunctions, most or all of which are caused by a lack of acidification must therefore employ a PtdIns(3,5)P -depen- PtdIns(3,5)P ? Do cells have one PtdIns(3,5)P effector pro- 2 2 2 dent pathway that needs none of the known Svp1p-related tein that contributes to multiple cell functions, or does proteins. PtdIns(3,5)P interact with several effectors, with each ful- filling a different function(s)? Protein sorting to the MVB. Cells that lack Fab1p or its With the aim of identifying PtdIns(3,5)P targets, we activator Vac14p do not correctly sort certain proteins into screened the EUROFAN deletion mutant collection for gene MVBs, and irreversible ubiquitination of cargo proteins cor- deletions that cause fab1D-like vacuole enlargement (Dove rects this defect (Odorizzi et al, 1998; Dove et al, 2002). et al, 2002). From this screen, YFR021w/SVP1 was particu- This process can be monitored by assessing traffic to the larly intriguing—it is very near YFR019w/FAB1 on chromo- vacuole of GFP-Phm5 (Dove et al, 2002). This normally goes some VI and all eukaryotes have homologues. into the lumen, but defective sorting mislocates it at the membrane. GFP-Phm5p sorting was normal in svp1D and PtdIns(3,5)P specificity of Svp1p and related proteins triple-deletion svp1D//hsv1D/hsv2D cells, but defective in That GFP-Svp1p localises to the vacuole in a FAB1-dependent fab1Dcells (Figure 7D)—so none of the Svp1p-like proteins manner suggested that Svp1p might be a PtdIns(3,5)P effec- contribute to this PtdIns(3,5)P -dependent process. tor. However, Svp1p and Fab1p also seem to interact directly (Georgakopoulos et al, 2001), so that might also contribute to the vacuole localisation. Others have also concluded that Discussion some Svp1p is localised to a punctate compartment near The first insight into the biology of PtdIns(3,5)P came with the vacuole (Barth et al, 2001). Very gentle lysis disrupted the the discovery that Fab1p is the PtdIns3P 5-kinase that makes vacuole localisation of GFP-Svp1p but not its punctuate this lipid (Cooke et al, 1998; Gary et al, 1998). Cells lacking localisation (Guan et al, 2001), and our evidence also indi- FAB1, or expressing a kinase-inactive version of Fab1p in a cates that different mechanisms regulate Svp1p targeting to fab1D background (F Cooke, unpublished), display a com- each compartment. plex spectrum of dysfunctions, including vacuole enlarge- Under approximately ‘physiological’ conditions of ionic 2 þ ment, mis-sorting of proteins into MVBs, heat-sensitive cell strength and Mg concentration, monomeric Svp1p is a &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1929 | | WD-40 motifs can act as lipid-binding modules SK Dove et al very selective and high-affinity PtdIns(3,5)P -binder, as are two related yeast proteins (Hsv1p and Hsv2p), the Drosophila homologue CG11975 and the human homologue hSvp1a. This suggests that Svp1p and its homologues constitute a substantial family of PtdIns(3,5)P specific effector proteins. These are the first proteins known to bind phosphoinositides through small basic amino-acid patches on a b-propeller structure, and so it is possible that a subset of the many proteins encoding b-propeller/WD-40 motifs may also bind to phosphoinositides. This localisation of the Svp1p PtdIns(3,5)P binding on its b-propeller also suggests a possible structural analogy with the recently discovered binding of a phosphothreonine-containing peptide to the eight-bladed b-propeller of Cdc4p (Orlicky et al, 2003). Cellular roles of Svp1p The normal yeast vacuole comprises an array of large orga- nelles that undergo homotypic fusion with other vacuole elements (Wickner, 2002) and heterotypic fusion with smal- ler organelles, including MVBs (Odorizzi et al, 1998). Following the recruitment of membrane by fusion, the va- cuole and MVB elements may normally re-segregate in a manner similar to that seen in cell-free studies with mamma- lian lysosome/late endosome hybrid organelles (Luzio et al, 2000) or a vesicular intermediate may be involved in vacuole to late endosome trafficking. It is likely that Svp1p is involved in a PtdIns(3,5)P -dependent prebudding step in this vacuole membrane segregation process, as suggested in the model in Figure 8A. When this retrograde trafficking step is blocked, as in fab1D and svp1D cells, far more vacuole membrane Figure 8 The involvement of Fab1p, PtdIns(3,5)P and down- accumulates than in normal cells and the multiple vacuole stream effector proteins in yeast cell functions. (A) Outline of the elements fuse (Yamamoto et al, 1995). cycle of vacuole membrane addition and retrieval for which PtdIns(3,5)P appears to be essential. It is not clear whether retro- Studies of trafficking of the v-SNARE, Vti1p, have con- grade vacuole-to-late-endosome trafficking occurs by re-segregation firmed a defect in this segregation/reformation process when of the vacuole and late endosome or by the traffic of a vesicular PtdIns(3,5)P production is impaired. Vti1p is involved in the intermediate between these structures. (B) A tentative synthesis of fusion of late endosomes/MVBs and the vacuole, and a how the actions of multiple PtdIns(3,5)P effector proteins may contribute to various cell functions (see Discussion). All of these, retrograde pathway normally recycles Vti1p to late endo- except the involvement of Svp1p in autophagy, require the presence somes. This is blocked in vac7D cells (Bryant et al, 1998), in cells of FAB1 and/or PtdIns(3,5)P . which lack the Fab1p activator Vac7p and make little PtdIns(3,5)P (Gary et al, 2002). Second mutations that restore PtdIns(3,5)P to normal levels rescue the vac7D action between Fab1p and Svp1p (Georgakopoulos et al, defects: for example, a suppressive mutation of FIG4, which 2001) suggests a possible mechanism for feedback regulation encodes a phosphoinositide phosphatase (Gary et al, 2002). of PtdIns(3,5)P synthesis. Our RS-ALP trafficking results show that svp1D cells pheno- copy this vac7D defect, almost certainly because Svp1p is the PtdIns(3,5)P effector required for this membrane recycling. Svp1p as one of several PtdIns(3,5)P effectors? 2 2 Our working hypothesis is that the Svp1p/PtdIns(3,5)P Taken with recent reports that Ent3p, Ent5p and Vps24p are complex participates in a specific interaction with some other effectors that contribute to the MVB trafficking functions of protein (or complex), and so directs budding from the PtdIns(3,5)P (Friant et al, 2003; Whitley et al, 2003), our vacuole surface. Folded as a b-propeller, Svp1p is well suited results support the idea that the complex fab1D phenotype is to form a platform for protein–protein interactions, probably caused by malfunctions in several, probably independent, of the peptide-in-groove type (ter Haar et al, 2000). Our PtdIns(3,5)P effector pathways. It is clear that neither Ent3p, autophagy studies make it clear that Svp1p also performs Ent5p nor Vps24p (Friant et al, 2003; Whitley et al, 2003), nor PtdIns(3,5)P -independent vacuole-related functions, and any of the Svp1p-related proteins, participates in the effects of involvement of the same binding partners in these func- PtdIns(3,5)P on vacuole acidification or heat tolerance. tions might constitute a mechanistic link between the Elsewhere, we shall describe Svp3p, an unrelated putative PtdIns(3,5)P -dependent and -independent functions of PtdIns(3,5)P effector that influences PtdIns(3,5)P metabo- 2 2 2 Svp1p (Barth et al, 2001; Guan et al, 2001). lism and appears to contribute to MVB sorting and to heat Since svp1D cells accumulate abnormally large amounts tolerance (SK Dove, unpublished; see Figures 7B and D). of PtdIns(3,5)P , it also seems likely that the Svp1p/ Other observations support the notion that PtdIns(3,5)P 2 2 PtdIns(3,5)P complex either restrains PtdIns(3,5)P synth- exerts its effects through multiple effector pathways. For 2 2 esis or activates PtdIns(3,5)P degradation. The known inter- example, low overexpression of the FAB1 gene in the 1930 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al et al, 1997). Mutants were sequenced and then moved into GST vac14-1 background restores vacuole acidification, but the expression vectors. vacuole remains enlarged. However, vacuole enlargement is only corrected if FAB1 is greatly overexpressed (Bonangelino Expression of GST-Svp1p and GST-Hsv2p et al, 2002; Dove et al, 2002). Similarly, the catalytic site The SVP1 and HSV2 ORFs were cloned into pGEX-6P-1 (Amersham- G2042V/G2045V mutant FAB1 normalises MVB sorting in fab1D Pharmacia) (with a rhinovirus-2C protease cleavage site between GST and the target protein) and pGSTag (Ron and Dressler, 1992). cells but does not correct the vacuole enlargement (Odorizzi Both plasmids were transformed into Escherichia coli BL21 and et al, 1998). expressed as follows: cells were grown in LB þ ampicillin (100 mg/ Figure 8B summarises our current understanding of ml) to an OD of 0.5–0.8, induced with IPTG (200–500 mM, 2–4 h, PtdIns(3,5)P effector pathways. It highlights the fact that 301C), harvested, washed in ice-cold PBS containing protease inhibitors (pepstatin A, 5 mg/ml; leupeptin 5 mg/ml; E-64, 3 mg/ml; none of the currently known PtdIns(3,5)P effectors mediates aprotinin, 5 mg/ml; PMSF, 100 mM), lysed (100 mg/ml lysozyme, PtdIns(3,5)P -dependent vacuole acidification, and therefore 41C, 30 min) and disrupted with glass beads in a BeadBeater suggests that at least one S. cerevisiae PtdIns(3,5)P effector (6  30 s, 41C). Triton X-100 (1%) supernatants from the disrupted still awaits discovery. Furthermore, Hsv1p and Hsv2p are cells (8000 g, 15 min; then 100 000 g, 1 h) were filtered (0.2 mm) and applied to glutathione–sepharose 4B beads (30 min, 41C), which likely to be PtdIns(3,5)P effectors, but their loss causes none were eluted following the manufacturer’s recommendations. of the known FAB1/PtdIns(3,5)P -dependent phenotypes—it Proteins were made 50% in ice-cold glycerol, snap-frozen and is therefore probable that undiscovered PtdIns(3,5)P -depen- stored at 801C. dent cellular processes remain to be found. Phosphoinositide dot blots SVP1 and HSV2 cloned into the pGSTag vector (with a PKA Materials and methods phosphorylation site in the GST linker) were expressed in E. coli 32 32 BL21 (see above) and P-labelled (Kavran et al, 1998). [ P]pro- Most materials were from sources defined previously, and methods teins were filtered (0.2 mm) and used to probe lipid ‘dot blots’ of for the growth of yeast, for FM4-64 vacuolar staining, for the GFP- serial two-fold dilutions of phosphoinositides (from 1000 to Phm5p MVB sorting assay, for quinacrine staining and for lipid 15.6 pmol, in CHCl :CH OH:H 0, 400:56:4, v/v/v) on Hybond C 3 3 2 have been described (Dove et al, 1997, 2002; Dove and Michell, membrane (BioRad). Blots were blocked (3 h, room temperature, 1999; McEwen et al, 1999). The source of all yeast strains used in 10 ml PBS, 0.5 mM MgCl , 5% ECL blocking reagent) and probed 6 32 this study is indicated in Table I. PtdIns4P and PtdIns5P were from with 1–2 10 dpm of [ P]protein (30 min, room temperature). Echelon Inc. (Salt Lake City, UT), and PtdIns(3,4)P , PtdIns(4,5)P , 2 2 Blots were extensively washed (PBS/0.5 mM MgCl ) and radio- PtdIns(3,4,5)P , and PtdIns3P were from Matreya Inc. (Pleasant activity detected by autoradiography. Concentrations of the lipid Gap, PA): all were dipalmitoyl. Dioleoyl-PtdCho (DOPC), dipalmi- stocks were quantified immediately before spotting by wet ashing toyl-PtdSer and PtdIns were from Sigma-Aldrich. Phosphoinositides and phosphate determination (Baginski et al, 1967). and Affigel-linked PtdIns(3,5)P were synthesised as described (Krugmann et al, 2002). For Biacore analysis, PtdIns(3,5)P was Protein binding to Affigel-linked PtdIns(3,5)P from CellSignals Inc. (Lexington, KY). GST-Svp1p (5 mM) was incubated (1 ml PBS containing 0.1% Triton X-100, 10 mM DTT and 50 mM PMSF) with 20ml of Affigel-20 beads Vacuole morphology screen covalently linked to PtdIns(3,5)P , in the presence of micellar This screen was as described (Dove et al, 2002). Subsequent dispersions of various phosphoinositides (10 mM) for 30–60 min at analysis of the identified genes included bioinformatic sifting to 41C. In 10 min or less, the beads were sedimented (12 000 g, 1 min) eliminate genes unrelated to vacuole function and to prioritise and washed in the above buffer (twice) and in 5 mM Hepes/KOH, genes with homologues in many organisms. pH 7.5. The remaining bead-bound protein was solubilised in 40 ml SDS–PAGE sample buffer and resolved on 10% SDS–PAGE minigels 0 0 (made with piperazine diacrylamide (BioRad) rather than N N - Plasmid construction methylene bis-acrylamide, to reduce the silver-staining back- GFP-Svp1p and GFP-Hsv2p were constructed by PCR amplification ground). Gels were fixed and stained (BioRad Silver Stain Plus). of the respective ORFs from yeast genomic DNA using the expand proof-reading DNA polymerase (Roche). The Met-regulated con- struct pUG36-SVP1 was created by ligating the SVP1 ORF cut with Biacore analysis of phosphoinositide binding EcoRI and HindIII into pUG36. pUG36-HSV2 was created by ligating This was carried out exactly as described (Yu and Lemmon, 2001). the complete HSV2 ORF, excised with BamHI and SalI, into pUG36. Positive controls were performed alongside Svp1p experiments, and Transformed yeast were grown to 1 10 cells/ml in SC-Ura-Met and included the PLCd1 PH domain (binds PtdIns(4,5)P ), the DAPP1 visualised as described (Dove et al, 2002). Mutations in the SVP1 PH domain (binds PtdIns(3,4)P ), the FAPP1 PH domain (binds gene were carried out using serial overlap extension PCR (Warrens PtdIns4P) and the Hrs1 FYVE domain (binds PtdIns3P). Lipid Table I Yeast strains used in this study Yeast strain Source Reference BY4742 mat a ura3D0 his3D1 leu2D0 lys2D0 EUROSCARF N/A BY4742 svp1HKANMX4 EUROSCARF N/A BY4742 svp1HKANMX4 hsv1HLEU2 hsv2HHIS3 This study N/A BY4742 hsv1HLEU2 hsv2HHIS3 D Alexandraki Georgakopoulos et al (2001) BY4742 hsv1HKANMX4 EUROSCARF N/A BY4742 hsv2HKANMX4 EUROSCARF N/A BY4742 fab1HKANMX4 EUROSCARF N/A BY4742 fab1HKANMX4 pho8HLEU2 This study N/A BY4742 hsv1HKANMX4 pho8HLEU2 This study N/A BY4742 hsv2HKANMX4 pho8HLEU2 This study N/A BY4742 svp1HKANMX4 pho8HLEU2 This study N/A BY4742 svp2HKANMX4 EUROSCARF N/A BY4742 svp3HKANMX4 EUROSCARF N/A NA: not applicable. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1931 | | WD-40 motifs can act as lipid-binding modules SK Dove et al surfaces were used within 8 h of generation, since signal strength 50 mM Tris–HCl, 5 mM MgCl , 1 mM PMSF and 1 mg/ml pepstatin A, began to decrease within B12 h. pH 9, cooled (15 min) and vortexed with glass beads (8  30 s, with GST was quantitatively removed from GST-Svp1p with PreScis- 30 s on ice between cycles). Lysates were centrifuged (10 000 g, sion protease. When gel-filtered on Superose-6 (Amersham-Phar- 15 min, 41C). The supernatant protein (5 mg) was incubated at 251C macia), the liberated Svp1p eluted as a 60–70 kDa monomer. Svp1p for 3 min in 1 ml of 10 mM p-nitrophenolphosphate, 0.5 M Tris and binding was measured by simultaneously passing it over a 2.5 mM MgCl , pH 8.8, with continuous monitoring of OD . 2 420 phosphoinositide-containing sensor surface and a control DOPC Relative rates of dephosphorylation rates were calculated for the sensor surface, with the control signal subtracted from that from the induced and uninduced cells. phosphoinositide surface. Binding data are plotted as per cent of maximum binding against protein concentration injected. K values Dilution assays for temperature sensitivity of growth were calculated as described (Yu and Lemmon, 2001). Yeast were diluted to 1 10 cells/ml, serially diluted four-fold, and 5 ml samples spotted on replicate plates. One was incubated at 231C Estimating K value and the other at 421C, until wild-type cells formed distinct colonies For Svp1p binding to PtdIns(3,5)P , PtdIns(3,4)P and 2 2 at all dilutions. PtdIns(4,5)P , data were fitted to the following equation: 0 1 ½Prot Assay for retrograde vacuole to late endosome trafficking Cells expressing RS-ALP were spheroplasted as previously de- @ A Percent maximal binding ¼ 100 þ Y ½Prot scribed (Urbanowski and Piper, 2001) and lysed in 300 mM sorbitol, þ 1 20 mM HEPES, pH 7.2, and 1 mM EDTA (HES buffer) containing a where [Prot] is the flowing protein concentration (assumed protease inhibitor cocktail (Complete tm, Boehringer Mannheim). unaffected by binding to the surface), K is the dissociation D Postnuclear supernatants were adjusted to 30% Optiprep and constant and Y corresponds to a residual or background signal. layered beneath 5 ml of HES buffer in an SW41 Beckman Fitting was performed using ORIGIN (MicroCal), with floating K D ultracentrifuge tube. Linear Optiprep gradients were overlaid and Y. (BioComp gradient mixer) and the samples were centrifuged (40 000 rpm, 18 h). Fractions were collected downwards from the [ H]inositol labelling and phosphoinositide analysis top. Proteins were separated by SDS–PAGE and immunoblotted Yeast were labelled in inositol-free media for 5–6 cell divisions, so using monoclonal anti-ALP and anti-Vph1p antibodies (Molecular that changes in [ H] would parallel changes in lipid concentration, Probes) and a polyclonal anti-Vps10p antiserum (Piper et al, 1995). and phosphoinositides were extracted and analysed as described (Cooke et al, 1998). Acknowledgements Assay of Pho8D60p maturation This used a method described by Noda et al (1995). PHO8 was We thank Drs Paul Whitley (University of Bath, UK), Gerald Hammond disrupted in appropriate strains using a pho8HLEU2 cassette, and (UCL, London), Despina Alexandraki (University of Crete), Lois the disruptants were transformed with a plasmid that overexpresses Weisman (Iowa) and Geraint Thomas (UCL, London) for valuable Pho8D60p (pTN3, a gift from Dr T Noda, National Institute for Basic discussions and reagents. This work was funded by the Royal Society Biology, Okazaki, Japan). These strains were grown to (SKD and RHM), the Wellcome Trust (SKD and RHM), the BBSRC (to B1 10 cells/ml, washed and either processed immediately or ABT and JT) and grant 5-ROI-GM56846 from the National Institutes of incubated in a nitrogen-free medium for 8 h at 301C, to induce Health (to MAL). SKD is a Royal Society University Research Fellow. autophagy and then processed. They were suspended in 1 ml of RHM is a Royal Society Research Professor. References Abeliovich H, Zhang C, Dunn Jr WA, Shokat KM, Klionsky DJ for its ability to regulate epidermal growth factor receptor degra- (2003) Chemical genetic analysis of Apg1 reveals a non-kinase dation. J Biol Chem 277: 48730–48736 role in the induction of autophagy. Mol Biol Cell 14: 477–490 Dove SK, Cooke FT, Douglas MR, Sayers LG, Parker PJ, Michell RH Baginski ES, Foa PP, Zak B (1967) Microdetermination of inorganic (1997) Osmotic stress activates phosphatidylinositol-3,5-bispho- phosphate, phospholipids, and total phosphate in biologic mate- sphate synthesis. Nature 390: 187–192 rials. Clin Chem 13: 326–332 Dove SK, McEwen RK, Mayes A, Hughes DC, Beggs JD, Michell RH Barth H, Meiling-Wesse K, Epple UD, Thumm M (2001) Autophagy (2002) Vac14 controls PtdIns(3,5)P synthesis and Fab1-depen- and the cytoplasm to vacuole targeting pathway both require dent protein trafficking to the multivesicular body. Curr Biol 12: Aut10p. FEBS Lett 508: 23–28 885–893 Barth H, Meiling-Wesse K, Epple UD, Thumm M (2002) Mai1p is Dove SK, Michell RH (1999) Analysis of the polyphosphorylated essential for maturation of proaminopeptidase I but not for inositol lipids of S. cerevisiae.In Signalling Research: A Practical autophagy. FEBS Lett 512: 173–179 Approach, Milligan G (ed) pp 255–261. Oxford University Press Bonangelino CJ, Catlett NL, Weisman LS (1997) Vac7p, a novel Ellson CD, Andrews S, Stephens LR, Hawkins PT (2002) The PX vacuolar protein, is required for normal vacuole inheritance and domain: a new phosphoinositide-binding module. J Cell Sci 115: morphology. Mol Cell Biol 17: 6847–6858 1099–1105 Bonangelino CJ, Nau JJ, Duex JE, Brinkman M, Wurmser AE, Ferguson KM, Kavran JM, Sankaran VG, Fournier E, Isakoff SJ, Gary JD, Emr SD, Weisman LS (2002) Osmotic stress-induced Skolnik EY, Lemmon MA (2000) Structural basis for discrimina- increase of phosphatidylinositol 3,5-bisphosphate requires tion of 3-phosphoinositides by pleckstrin homology domains. Mol Vac14p, an activator of the lipid kinase Fab1p. J Cell Biol 156: Cell 6: 373–384 1015–1028 Friant S, Pecheur EI, Eugster A, Michel F, Lefkir Y, Nourrisson Bryant NJ, Piper RC, Weisman LS, Stevens TH (1998) Retrograde D, Letourneur F (2003) Ent3p is a PtdIns(3,5)P effector traffic out of the yeast vacuole to the TGN occurs via the required for protein sorting to the multivesicular body. Dev Cell prevacuolar/endosomal compartment. J Cell Biol 142: 651–663 5: 499–511 Cooke FT, Dove SK, McEwen RK, Painter G, Holmes AB, Hall MN, Gary JD, Sato TK, Stefan CJ, Bonangelino CJ, Weisman LS, Emr SD Michell RH, Parker PJ (1998) The stress-activated phosphatidyli- (2002) Regulation of Fab1 phosphatidylinositol 3-phosphate 5- nositol 3-phosphate 5-kinase Fab1p is essential for vacuole func- kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide tion in S. cerevisiae. Curr Biol 8: 1219–1222 phosphatase family member. Mol Biol Cell 13: 1238–1251 Cozier GE, Carlton J, McGregor AH, Gleeson PA, Teasdale RD, Gary JD, Wurmser AE, Bonangelino CJ, Weisman LS, Emr SD Mellor H, Cullen PJ (2002) The phox homology (PX) domain- (1998) Fab1p is essential for PtdIns(3)P 5-kinase activity and dependent, 3-phosphoinositide-mediated association of sorting the maintenance of vacuolar size and membrane homeostasis. nexin-1 with an early sorting endosomal compartment is required J Cell Biol 143: 65–79 1932 The EMBO Journal VOL 23 NO 9 2004 &2004 European Molecular Biology Organization | | WD-40 motifs can act as lipid-binding modules SK Dove et al Georgakopoulos T, Koutroubas G, Vakonakis I, Tzermia M, Noda T, Suzuki K, Ohsumi Y (2002) Yeast autophagosomes: Prokova V, Voutsina A, Alexandraki D (2001) Functional analysis de novo formation of a membrane structure. Trends Cell Biol 12: of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w 231–235 ORF family suggests relations to mitochondrial/peroxisomal Odorizzi G, Babst M, Emr SD (1998) Fab1p PtdIns(3)P 5-kinase functions and amino acid signalling pathways. Yeast 18: function essential for protein sorting in the multivesicular body. 1155–1171 Cell 95: 847–858 Gillooly DJ, Simonsen A, Stenmark H (2001) Cellular functions of Orlicky S, Tang X, Willems A, Tyers M, Sicheri F (2003) Structural phosphatidylinositol 3-phosphate and FYVE domain proteins. basis for phosphodependent substrate selection and orientation Biochem J 355: 249–258 by the SCFCdc4 ubiquitin ligase. Cell 112: 243–256 Guan J, Stromhaug PE, George MD, Habibzadegah-Tari P, Bevan A, Piper RC, Cooper AA, Yang H, Stevens TH (1995) VPS27 Dunn Jr WA, Klionsky DJ (2001) Cvt18/Gsa12 is required for controls vacuolar and endocytic traffic through a prevacuolar cytoplasm-to-vacuole transport, pexophagy, and autophagy in compartment in Saccharomyces cerevisiae. J Cell Biol 131: Saccharomyces cerevisiae and Pichia pastoris. Mol Biol Cell 12: 603–617 3821–3838 Rebecchi MJ, Pentyala SN (2000) Structure, function, and control Harding TM, Hefner-Gravink A, Thumm M, Klionsky DJ (1996) of phosphoinositide-specific phospholipase C. Physiol Rev 80: Genetic and phenotypic overlap between autophagy and the 1291–1335 cytoplasm to vacuole protein targeting pathway. J Biol Chem Ron D, Dressler H (1992) pGSTag—a versatile bacterial expression 271: 17621–17624 plasmid for enzymatic labeling of recombinant proteins. Huang WP, Klionsky DJ (2002) Autophagy in yeast: a review of the Biotechniques 13: 866–869 molecular machinery. Cell Struct Funct 27: 409–420 Sankaran VG, Klein DE, Sachdeva MM, Lemmon MA (2001) High- Ikonomov OC, Sbrissa D, Shisheva A (2001) Mammalian cell affinity binding of a FYVE domain to phosphatidylinositol 3- morphology and endocytic membrane homeostasis require en- phosphate requires intact phospholipid but not FYVE domain zymatically active phosphoinositide 5-kinase PIKfyve. J Biol oligomerization. Biochemistry 40: 8581–8587 Chem 276: 26141–26147 Sondek J, Bohm A, Lambright DG, Hamm HE, Sigler PB (1996) Kavran JM, Klein DE, Lee A, Falasca M, Isakoff SJ, Skolnik EY, Crystal structure of a G-protein beta gamma dimer at 2.1 A Lemmon MA (1998) Specificity and promiscuity in phosphoinosi- resolution. Nature 379: 369–374 tide binding by pleckstrin homology domains. J Biol Chem 273: Stenmark H, Aasland R (1999) FYVE-finger proteins—effectors of 30497–30508 an inositol lipid. J Cell Sci 112: 4175–4183 Kelley LA, MacCallum RM, Sternberg MJ (2000) Enhanced genome ter Haar E, Harrison SC, Kirchhausen T (2000) Peptide-in-groove annotation using structural profiles in the program 3D-PSSM. interactions link target proteins to the beta-propeller of clathrin. J Mol Biol 299: 499–520 Proc Natl Acad Sci USA 97: 1096–1100 Klionsky DJ, Cregg JM, Dunn Jr WA, Emr SD, Sakai Y, Sandoval IV, Urbanowski JL, Piper RC (2001) Ubiquitin sorts proteins into the Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y intralumenal degradative compartment of the late-endosome/ (2003) A unified nomenclature for yeast autophagy-related vacuole. Traffic 2: 622–630 genes. Dev Cell 5: 539–545 Walker DM, Urbe S, Dove SK, Tenza D, Raposo G, Clague MJ (2001) Krugmann S, Anderson KE, Ridley SH, Risso N, McGregor A, Characterization of MTMR3. An inositol lipid 3-phosphatase with Coadwell J, Davidson K, Eguinoa A, Ellson CD, Lipp P, novel substrate specificity. Curr Biol 11: 1600–1605 Manifava M, Ktistakis N, Painter G, Thuring JW, Cooper MA, Warrens AN, Jones MD, Lechler RI (1997) Splicing by overlap Lim ZY, Holmes AB, Dove SK, Michell RH, Grewal A, Nazarian A, extension by PCR using asymmetric amplification: an improved Erdjument-Bromage H, Tempst P, Stephens LR, Hawkins PT technique for the generation of hybrid proteins of immunological (2002) Identification of ARAP3, a novel PI3K effector regulating interest. Gene 186: 29–35 both Arf and Rho GTPases, by selective capture on phosphoinosi- Whiteford CC, Brearley CA, Ulug ET (1997) Phosphatidylinositol tide affinity matrices. Mol Cell 9: 95–108 3,5-bisphosphate defines a novel PI 3-kinase pathway in resting Lemmon MA, Ferguson KM (2000) Signal-dependent membrane tar- mouse fibroblasts. Biochem J 323: 597–601 geting by pleckstrin homology (PH) domains. Biochem J 350: 1–18 Whitley P, Reaves BJ, Hashimoto M, Riley AM, Potter BV, Luzio JP, Rous BA, Bright NA, Pryor PR, Mullock BM, Piper RC Holman GD (2003) Identification of mammalian Vps24p as an (2000) Lysosome–endosome fusion and lysosome biogenesis. effector of phosphatidylinositol 3,5-bisphosphate-dependent J Cell Sci 113: 1515–1524 endosome compartmentalization. J Biol Chem 278: 38786–38795 Matsuura A, Tsukada M, Wada Y, Ohsumi Y (1997) Apg1p, a novel Wickner W (2002) Yeast vacuoles and membrane fusion pathways. protein kinase required for the autophagic process in EMBO J 21: 1241–1247 Saccharomyces cerevisiae. Gene 192: 245–250 Xu Y, Seet LF, Hanson B, Hong W (2001) The Phox homology (PX) McEwen RK, Dove SK, Cooke FT, Painter GF, Holmes AB, Shisheva domain, a new player in phosphoinositide signalling. Biochem J A, Ohya Y, Parker PJ, Michell RH (1999) Complementation 360: 513–530 analysis in PtdInsP kinase-deficient yeast mutants demonstrates Yamamoto A, DeWald DB, Boronenkov IV, Anderson RA, Emr SD, that Schizosaccharomyces pombe and murine Fab1p homologues Koshland D (1995) Novel PI(4)P 5-kinase homologue, Fab1p, are phosphatidylinositol 3-phosphate 5-kinases. J Biol Chem 274: essential for normal vacuole function and morphology in yeast. 33905–33912 Mol Biol Cell 6: 525–539 Noda T, Matsuura A, Wada Y, Ohsumi Y (1995) Novel system for Yu JW, Lemmon MA (2001) All phox homology (PX) domains from monitoring autophagy in the yeast Saccharomyces cerevisiae. Saccharomyces cerevisiae specifically recognize phosphatidylino- Biochem Biophys Res Commun 210: 126–132 sitol 3-phosphate. J Biol Chem 276: 44179–44184 &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 9 2004 1933 | |

Journal

The EMBO Journal – Springer Journals

Published: May 5, 2004

Keywords: ATG18; CVT18; AUT10; lysosome; phosphoinositide

There are no references for this article.