TY - JOUR
AU - Hara-Nishimura, Ikuko
AB - Abstract Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) as larger precursors and are sorted to protein storage vacuoles, where they are converted into the mature forms. We report here an Arabidopsis mutant, maigo 1 ( mag1 ), which abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag1 seeds mis-sort storage proteins by secreting them from cells. mag1 seeds have smaller protein storage vacuoles in the seeds than do wild-type seeds. The MAG1 gene encodes a homolog of the yeast ( Saccharomyces cerevisiae ) protein VPS29. VPS29 is a component of a retromer complex for recycling a vacuolar sorting receptor VPS10 from the pre-vacuolar compartment to the Golgi complex. Our findings suggest that MAG1/AtVPS29 protein is involved in recycling a plant receptor for the efficient sorting of seed storage proteins. The mag1 mutant exhibits a dwarf phenotype. A plant retromer complex plays a significant role in plant growth and development. Introduction The seeds of higher plants accumulate large quantities of storage proteins, such as globulins and albumins. During seed maturation, the precursors of storage proteins are synthesized on rough endoplasmic reticulum (ER) and are sorted to protein storage vacuoles (PSVs) by vesicle-mediated machinery (Jolliffe et al. 2005 , Robinson et al. 2005 ), where they are converted into the mature forms (Shimada et al. 2003b ). Multiple pathways have been reported in the transport of storage proteins from ER to PSVs. Pea globulins are transported via Golgi complex where dense vesicles are formed (Hillmer et al. 2001 ). Storage proteins of pumpkin, in contrast, are transported to PSVs in a Golgi-independent manner by precursor-accumulating (PAC) vesicles (Hara-Nishimura et al. 1998 ). Seven putative vacuolar sorting receptors (AtVSRs) were identified in the Arabidopsis genome (Masclaux et al. 2005 ). Previously, we reported that among these receptors, AtVSR1/AtELP is responsible for the sorting of storage proteins in Arabidopsis seeds (Shimada et al. 2003a ). The atvsr1 mutant mis-sorts storage proteins by secreting them from cells, resulting in the abnormal accumulation of the precursors of 12S globulin and 2S albumin in the seeds. We also found a pumpkin VSR, PV72, on the membrane of PAC vesicles (Shimada et al. 1997 ), and PV72 binds to the precursor of 2S albumin, a major storage protein in pumpkin (Shimada et al. 2002 , Watanabe et al. 2002 ). The vacuolar targeting signals of pro2S albumin and proricin of castor bean were reported to bind to VSR-like proteins (Jolliffe et al. 2004 ). These findings suggest a VSR-mediated system function in transport of seed storage proteins (Hara-Nishimura et al. 2004 ). Another possible receptor (RMR, receptor homology region transmembrane domain ring H2 motif protein) was reported for PSV-destined proteins (Jiang et al. 2000 , Park et al. 2005 ). Further analysis with an RMR-deficient mutant is necessary to demonstrate the function of RMR as a sorting receptor. Delivery of vacuolar proteins synthesized on rough ER to vacuoles requires many factors known as vacuolar protein sorting (VPS) in yeast (Bowers and Stevens 2005 ). A vacuolar sorting receptor VPS10 sorts precursors of vacuolar hydrolytic enzymes, such as carboxypeptidase Y (CPY), from the late Golgi complex to the pre-vacuolar compartment (PVC) (Marcusson et al. 1994 ). After releasing the precursors at the PVC, VPS10 is recycled from the PVC to the late Golgi complex to mediate further rounds of sorting. For this VPS10 recycling, yeast has machinery called a retromer (Seaman et al. 1997 ). The retromer complex is comprised of five cytosolic or peripheral membrane proteins termed VPS5, VPS17, VPS26, VPS29 and VPS35 (Seaman et al. 1998 ). Retromer components are highly conserved among various eukaryotes including mammals and plants. Human orthologs of VPS35, VPS29 and VPS26 assemble into multimeric complexes (Haft et al. 2000 ). Recently, human VPS35 was reported to interact with the cytosolic domain of the cation-independent mannose 6-phosphate receptor (Arighi et al. 2004 ). The Arabidopsis genome contains several genes that encode putative components of the retromer complex (Oliviusson et al. 2006 ). However, the exact functions of the gene products remain to be determined. In this study, we screened for Arabidopsis mutants that have a defect in sorting of storage proteins. Here we show that deficiency of the Arabidopsis homolog of VSP29 (AtVPS29) causes mis-sorting of storage proteins, resulting in abnormal accumulation of precursors of the storage proteins in dry seeds. Our findings suggest that AtVPS29 is involved in recycling of AtVSR1, which sorts the precursors of storage proteins to PSVs during seed maturation. Results and Discussion Screening for Arabidopsis mutants that accumulate storage protein precursors in dry seeds To understand better the transport mechanism of seed storage proteins, we attempted to isolate Arabidopsis mutants that abnormally accumulated the precursors of storage proteins in dry seeds. We obtained 28,000 T-DNA-tagged lines of Arabidopsis and subjected the extracts from dry seeds of each line to immunoblots with specific antibodies against two major storage proteins, 12S globulin and 2S albumin. Finally, we succeeded in isolating eight mutant lines. We designated these lines maigo ( mag ) mutants (maigo means a stray or lost child in Japanese), because a significant number of storage proteins were not transported to their destination PSVs in these mutants. Arabidopsis wild-type seeds accumulated the α- and β-subunits of the 12S globulins and the large and small subunits of the 2S albumins ( Fig. 1 , WT). On the other hand, mag1-1 seeds accumulated abnormally high levels of the precursors of storage proteins, pro12S globulins (p12S) and pro2S albumins (p2S) ( Fig. 1 , mag1-1 ). The N-terminal sequences of the 49 and 54 kDa proteins found in mag1-1 seeds were determined to be XQREAPFPNAXH (X = undetermined amino acid) and RQSLGVPPQLQN, respectively. They are consistent with the sequences immediately following the co-translational cleavage sites for the signal peptides of 12S globulins, At12S2 and At12S1, respectively. The molecular masses of the two proteins, 49 and 54 kDa, confirmed that they are the proprotein precursors of the 12S globulins. Similarly the 17 kDa protein accumulated in the mag1-1 seeds appeared to be the proprotein precursor of 2S albumin. The abnormal accumulation of the precursor proteins suggests that mag1-1 has a defect in the intracellular transport of storage proteins. Fig. 1 View largeDownload slide The mag1-1 mutant accumulates the precursors of seed storage proteins in dry seeds. (A) Immunoblot analysis of the dry seeds (four grains) of the wild-type (WT) and mag1-1 mutant with anti-12S globulin (anti-12S) and anti-2S albumin (anti-2S) antibodies. (B) Protein profiles of the dry seeds of the wild-type (WT) and mag1-1 mutant. The mag1-1 seeds accumulated large amounts of the precursors of 12S globulin (p12S) and 2S albumin (p2S), whereas wild-type seeds accumulated only the mature forms of 12S globulin (12S) and 2S albumin (2S). α and β, 12S globulin subunits; L and S, 2S albumin subunits. The numbers indicate molecular masses of the precursors in kDa. Fig. 1 View largeDownload slide The mag1-1 mutant accumulates the precursors of seed storage proteins in dry seeds. (A) Immunoblot analysis of the dry seeds (four grains) of the wild-type (WT) and mag1-1 mutant with anti-12S globulin (anti-12S) and anti-2S albumin (anti-2S) antibodies. (B) Protein profiles of the dry seeds of the wild-type (WT) and mag1-1 mutant. The mag1-1 seeds accumulated large amounts of the precursors of 12S globulin (p12S) and 2S albumin (p2S), whereas wild-type seeds accumulated only the mature forms of 12S globulin (12S) and 2S albumin (2S). α and β, 12S globulin subunits; L and S, 2S albumin subunits. The numbers indicate molecular masses of the precursors in kDa. mag1 secretes the storage proteins from seed cells Storage proteins are accumulated in PSVs of seed cells. Fig. 2 A shows the autofluorescent PSVs of the wild-type seeds surrounded with non-fluorescing cell walls. An electron micrograph shows the ultrastructure of seed cells that contain electron-dense PSVs and electron-lucent lipid bodies ( Fig. 2 B). Fluorescence and electron microscopy revealed three types of abnormality in mag1-1 seeds. The first abnormality is the small size of the PSVs ( Fig. 2 C, D). The mag1-1 seeds have numerous PSVs in the cells and the PSVs are much smaller than those of the wild-type seeds. This finding suggests that the MAG1 gene is involved in the biogenesis of PSVs. Fig. 2 View largeDownload slide The mag1-1 mutant mis-sorts storage proteins by secreting them from cells. Auto-fluorescence of PSVs in the dry seeds of the wild-type (A) and the mag1-1 mutant (C). mag1-1 seeds have smaller PSV than wild-type seeds. Ultrastructures of seed cells of the wild-type (B) and mag1-1 mutant (D–F). The extracellular space of the mag1-1 seeds was abnormally filled with electron-dense material (arrowheads in D and E). Immunogold analysis with anti-12S globulin antibody (E). Immunogold particles were distributed in the PSVs and the electron-dense extracellular space of the mag1-1 seeds. Some parts of the cell wall of mag1-1 mutant were occasionally enlarged and formed abnormal structures (arrowheads in F ). CW, cell wall. Scale bars: A and B = 10 μm; C and D = 5 μm; E and F = 1 μm. Fig. 2 View largeDownload slide The mag1-1 mutant mis-sorts storage proteins by secreting them from cells. Auto-fluorescence of PSVs in the dry seeds of the wild-type (A) and the mag1-1 mutant (C). mag1-1 seeds have smaller PSV than wild-type seeds. Ultrastructures of seed cells of the wild-type (B) and mag1-1 mutant (D–F). The extracellular space of the mag1-1 seeds was abnormally filled with electron-dense material (arrowheads in D and E). Immunogold analysis with anti-12S globulin antibody (E). Immunogold particles were distributed in the PSVs and the electron-dense extracellular space of the mag1-1 seeds. Some parts of the cell wall of mag1-1 mutant were occasionally enlarged and formed abnormal structures (arrowheads in F ). CW, cell wall. Scale bars: A and B = 10 μm; C and D = 5 μm; E and F = 1 μm. The second abnormality is that the extracellular space of the mag1-1 seeds is filled with electron-dense material ( Fig. 2 D, E). Immunogold staining indicates that the electron-dense material contains high concentrations of 12S globulin ( Fig. 2 E) and 2S albumin (data not shown). No electron-dense material was found in the extracellular space of wild-type seeds ( Fig. 2 B). The storage proteins were distributed both in the PSVs and in the electron-dense extracellular space of the mag1-1 seeds, whereas they were deposited only in the PSVs of wild-type seeds. These observations indicate that the mag1-1 mutant mis-sorts the storage proteins by secreting them from cells and that MAG1 plays an important role in the sorting of both 12S globulin and 2S albumin to PSVs during seed development. The third abnormality is deformity of cell walls. Some parts of the cell wall of the mag1-1 mutant were occasionally enlarged and formed abnormal structures ( Fig. 2 F). Similar structures were also observed in atvsr1-1 seeds (data not shown), although the frequency was lower in atvsr1-1 than in mag1-1 seeds. The origin of the abnormal structures is unknown, but they may be caused by abnormal secretion of storage proteins. Originally, ‘ mag mutants’ referred to mutants that have a defect in the transport of storage proteins in seeds. However, mag1 plants also have abnormal phenotypes. The mag1-1 seeds are morphologically similar to wild-type seeds and they grow normally at the initial step of seed germination. Afterwards, however, the mag1 plants grow poorly and have small rosette leaves with short petioles ( Fig. 3 A). The plant height of mag1-1 is approximately one-third of that of the wild-type ( Fig. 3 B). These abnormalities indicate that the MAG1 protein affects the development of vegetative organs. Another mutant allele mag1-2 exhibits more severe phenotypes than does mag1-1 ( Fig. 3 ) and produces only a few seeds. mag1-2 is a null allele, while mag1-1 is not a null allele (discussed below). Fig. 3 View largeDownload slide The mag1 mutants exhibited the dwarf phenotype. (A) Twenty-seven-day-old plants of wild type (WT), mag1-1 , mag1-1 transformed with the At3g47810 gene ( mag1-1/MAG1 ), and mag1-2 . (B) Forty-day-old plants of wild type (WT), mag1-1 , mag1-1 transformed with the At3g47810 gene ( mag1-1/MAG1 ), and mag1-2 . Scale bars = 2 cm. Fig. 3 View largeDownload slide The mag1 mutants exhibited the dwarf phenotype. (A) Twenty-seven-day-old plants of wild type (WT), mag1-1 , mag1-1 transformed with the At3g47810 gene ( mag1-1/MAG1 ), and mag1-2 . (B) Forty-day-old plants of wild type (WT), mag1-1 , mag1-1 transformed with the At3g47810 gene ( mag1-1/MAG1 ), and mag1-2 . Scale bars = 2 cm. A defect of the At3g47810 gene is responsible for mag1 phenotypes To identify the MAG1 gene, a genomic sequence adjacent to the T-DNA insert of mag1 was recovered by thermal asymmetric interlaced PCR (TAIL-PCR). T-DNA was inserted at the 3′-untranslated region (57 bases downstream of the stop codon) of the At3g47810 gene ( Fig. 4 A). Levels of the At3g47810 transcript in mag1-1 were determined by reverse transcription with either an oligo(dT) primer or random primers followed by PCR with a specific primer set. The result with an oligo(dT) primer showed that the steady-state level of At3g47810 mRNA was significantly lower in mag1-1 than in wild-type ( Fig. 4 B, oligo dT). Real-time reverse transcription–PCR (RT–PCR) revealed an 8.4-fold reduction of the mRNA level in mag1-1 compared with the wild type (data not shown). These results indicate that the T-DNA insertion does not knock-out the At3g47810 gene. The result with random primers showed no significant difference between mag1 and the wild type ( Fig. 4 B, random). These results suggest that the insertion of T-DNA strongly affects the processing (polyadenylation) of the At3g47810 transcript. Fig. 4 View largeDownload slide Identification and expression analysis of the MAG1 gene. (A) A schematic representation of the MAG1 gene (At3g47810) and the positions of the T-DNA insertions in the mag1-1 and mag1-2 alleles. Boxes represent exons, solid lines represent introns, and dotted lines represent upstream and downstream regions of the At3g47810 transcript. Gray boxes represent protein-coding regions and open boxes represent untranslated regions. F1 and F2, a primer set used for PCR in B and C. (B) RT–PCR showing the At3g47810 transcript levels of duplicate samples from wild-type (WT) and mag1-1 . Oligo(dT) or random primers were used for reverse transcription with total RNAs from rosette leaves. The Actin2 gene was used as a control. (C) RT–PCR of wild type (WT), mag1-2 (heterozygous) and mag1-2 (homozygous). Random primers were used for reverse transcription with total RNAs from rosette leaves. The Actin2 gene was used as a control. Fig. 4 View largeDownload slide Identification and expression analysis of the MAG1 gene. (A) A schematic representation of the MAG1 gene (At3g47810) and the positions of the T-DNA insertions in the mag1-1 and mag1-2 alleles. Boxes represent exons, solid lines represent introns, and dotted lines represent upstream and downstream regions of the At3g47810 transcript. Gray boxes represent protein-coding regions and open boxes represent untranslated regions. F1 and F2, a primer set used for PCR in B and C. (B) RT–PCR showing the At3g47810 transcript levels of duplicate samples from wild-type (WT) and mag1-1 . Oligo(dT) or random primers were used for reverse transcription with total RNAs from rosette leaves. The Actin2 gene was used as a control. (C) RT–PCR of wild type (WT), mag1-2 (heterozygous) and mag1-2 (homozygous). Random primers were used for reverse transcription with total RNAs from rosette leaves. The Actin2 gene was used as a control. To determine whether the mutation of At3g47810 is responsible for the mag1 phenotypes, we complemented the mag1-1 mutant with a 4,317 bp genomic DNA fragment containing the wild-type At3g47810 gene. All transgenic plants subsequently generated using this construct accumulated no precursors of seed storage proteins in the seed ( Fig. 5 , mag1-1 / MAG1 ). These complemented transgenic lines were of a wild-type phenotype in appearance ( Fig. 3 , mag1-1 / MAG1 ). To characterize the knock-out phenotype of the At3g47810 gene, we established another allele, mag1-2 , which contained a T-DNA insertion at the third intron of the At3g47810 gene. mag1-2 homozygous plants produced no amplified fragment from the At3g47810 transcript by RT–PCR ( Fig. 4 C), indicating that mag1-2 is a null allele. The total seed protein level of the mag1-2 mutant (5.3 ± 1.0 μg per seed) was similar to that of the wild type (5.9 ± 0.5 μg per seed), but was lower than that of the mag1-1 mutant (8.0 ± 0.2 μg per seed). Fig. 5 shows that mag1-2 seeds accumulate the precursors of storage proteins, both 12S globulins and 2S albumins, as do mag1-1 seeds. These results clearly demonstrate that the defect of the At3g47810 gene is responsible for the mag1 phenotypes. Fig. 5 View largeDownload slide Seed protein profiles of mag1 alleles. Four dry seeds of each of the wild type (WT), the mag1-1 mutant, the mag1-2 mutant and the mag1-1 mutant transformed with the wild-type At3g47810 gene ( mag1-1/MAG1 ) were subjected to SDS–PAGE followed by Coomassie blue staining. p12S, the precursors of 12S globulin; p2S, the precursors of 2S albumin; 12S-α and 12S-β, 12S globulin subunits; L and S, 2S albumin subunits. Fig. 5 View largeDownload slide Seed protein profiles of mag1 alleles. Four dry seeds of each of the wild type (WT), the mag1-1 mutant, the mag1-2 mutant and the mag1-1 mutant transformed with the wild-type At3g47810 gene ( mag1-1/MAG1 ) were subjected to SDS–PAGE followed by Coomassie blue staining. p12S, the precursors of 12S globulin; p2S, the precursors of 2S albumin; 12S-α and 12S-β, 12S globulin subunits; L and S, 2S albumin subunits. The MAG1 gene encodes AtVPS29, a putative component of a retromer complex The At3g47810 gene encodes a 190 amino acid protein, which shows a significant similarity to VPS29 proteins from various eukaryotes including human (63% identity), fruit fly (59% identity) and fission yeast (45% identity) ( Fig. 6 ). The VPS29 gene was originally identified in budding yeast, Saccharomyces cerevisiae (Seaman et al. 1997 ). VPS29 along with four other proteins (VPS5, VPS17, VPS26 and VPS35) forms a retromer complex that mediates the VPS10 recycling. Defects in these factors resulted in the secretion of proCPY to the culture medium (Paravicini et al. 1992 , Bachhawat et al. 1994 , Horazdovsky et al. 1997 ). The specific function of VPS29 within the retromer complex is not known. Recently, the crystal structures of mammalian VPS29s were reported (Collins et al. 2005 , Wang et al. 2005 ). VPS29 has structural similarity to proteins of the phosphoesterase/nuclease family. Fig. 6 View largeDownload slide Sequence alignment of VPS29 homologs. Conserved amino acids are highlighted in black. The accession numbers are as follows: MAG1 (AtVPS29), CAB41864; HsVPS29 (human), Q9UBQ0; DmVPS29 (fruit fly), AAF51410; SpVPS29 (fission yeast) and CAB52425. Fig. 6 View largeDownload slide Sequence alignment of VPS29 homologs. Conserved amino acids are highlighted in black. The accession numbers are as follows: MAG1 (AtVPS29), CAB41864; HsVPS29 (human), Q9UBQ0; DmVPS29 (fruit fly), AAF51410; SpVPS29 (fission yeast) and CAB52425. All the members of putative retromer components were found in the Arabidopsis genome: At5g06140, At5g07120 and At5g58840 (which belong to the VPS5/17 family), At4g27690 and At5g53530 (the VPS26 family), At3g47810 (the VPS29 family), and At1g75850, At2g17790 and At3g51310 (the VPS35 family). This suggests that the products of these genes form a retromer complex to function in Arabidopsis cells. Very recently, plant retromers were characterized in Arabidopsis and tobacco BY-2 cells (Oliviusson et al. 2006 ). We investigated the effect of AtVPS29 deficiency on the other members of the retromer. The levels of AtVPS35 protein were lower in mag1-1 and mag1-2 leaves, in which no MAG1/AtVPS29 protein was detected, than in wild-type leaves ( Fig. 7 ). This result suggests that MAG1/AtVPS29 and AtVPS35 proteins function cooperatively in plant cells and a deficiency of MAG1/AtVPS29 protein influences the stability of AtVPS35 protein. Fig. 7 View largeDownload slide A deficiency of MAG1/AtVPS29 protein influences the level of AtVPS35 protein. Total extracts from the rosette leaves of the wild type (WT), mag1-1 and mag1-2 were subjected to SDS–PAGE followed by Coomassie blue staining (CBB) or immunoblot with either anti-AtVPS29 antibody or anti-AtVPS35 antibody. Specific signals for AtVPS29 and AtVPS35 are indicated by arrowheads. In association with the striking reductions of the AtVPS29 level in the mag1 leaves, the level of AtVPS35 was reduced. Fig. 7 View largeDownload slide A deficiency of MAG1/AtVPS29 protein influences the level of AtVPS35 protein. Total extracts from the rosette leaves of the wild type (WT), mag1-1 and mag1-2 were subjected to SDS–PAGE followed by Coomassie blue staining (CBB) or immunoblot with either anti-AtVPS29 antibody or anti-AtVPS35 antibody. Specific signals for AtVPS29 and AtVPS35 are indicated by arrowheads. In association with the striking reductions of the AtVPS29 level in the mag1 leaves, the level of AtVPS35 was reduced. Physiological functions of MAG1 in seeds and vegetative tissues Recently, it was reported that treatment of plant cells with wortmannin, an inhibitor of phosphatidylinositol 3-kinase activity, inhibited efficient recycling of the receptor and caused the secretion of vacuolar proteins (daSilva et al. 2005 ). Oliviusson et al. ( 2006 ) reported that the plant retromer was localized to pre-vacuolar compartments together with VSRs, and their data implied that VPS35 interacts with VSRs. These findings suggest that recycling of the VSR by the retromer is necessary for efficient transport of vacuolar proteins in plants. All the phenotypes of mag1 seeds are similar to those of atvsr1 seeds that we previously reported (Shimada et al. 2003a ). Namely, atvsr1 seeds mis-sort storage proteins, accumulate storage protein precursors and have smaller PSVs. Taken together, our results suggest that AtVPS29 functions in retrograde transport of AtVSR1 for the efficient sorting of storage proteins in Arabidopsis seeds. In addition to the accumulation of storage protein precursors in seeds, mag1 plants also exhibit a severe phenotype during vegetative growth ( Fig. 3 ). Among the Arabidopsis retromer components, MAG1/AtVPS29 is encoded by a single-copy gene (At3g47810). DNA microarray analysis revealed that the At3g47810 gene is ubiquitously expressed in various organs including leaves, roots, flowers and developing seeds (Schmid et al. 2005 ). Thus, the MAG1/AtVPS29 gene may play an indispensable role in various tissues of Arabidopsis . It is possible that MAG1/AtVPS29 is also involved in recycling of AtVSRs for vacuolar proteins such as acid hydrolases that function in vegetative tissues. Another possibility is that MAG1/AtVPS29 is required for recycling of unknown membrane protein(s) necessary for the vegetative growth. Our results indicate that a plant retromer complex plays a significant role in plant growth and development. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) were used as the wild-type plants. Seeds of Arabidopsis were surface sterilized and then sown on soil or onto 0.5% Gellan Gum (Wako, Tokyo, Japan) that contained 1% sucrose and Murasige–Skoog medium. After a 3 d incubation at 4°C to break seed dormancy, Arabidopsis plants were grown side by side at 22°C under continuous light. After incubation for 20 d on the plates, plants were transferred to soil for further incubation. Isolation of Arabidopsis mutants that accumulated precursors of 12S globulins and 2S albumins We screened Arabidopsis T-DNA tagged lines as descried previously (Shimada et al. 2003b ). Dry seeds (0.1 mg) from the mixture of 1–20 lines were subjected to SDS–PAGE and then to immunoblot with anti-12S globulin and anti-2S albumin to detect the storage protein precursors. We isolated eight lines ( mag mutants) that abnormally accumulated the precursors of storage proteins. We report here one of these mutants, mag1-1 , which is derived from the pool of T-DNA tagged lines that we prepared with Ti plasmid pBI121. A backcross of the mag1-1 mutant with the wild-type plant revealed that the mag1 mutation is recessive. T-DNA homozygous plants exhibited mutant phenotypes, suggesting that the mutation is influenced by the T-DNA insertion. The mag1-2 (G037C03) mutant allele was obtained from the GABI-Kat T-DNA mutant population. To identify the MAG1 gene, we amplified the flanking sequence of T-DNA by TAIL-PCR (Liu et al. 1995 ) using a border sequence of T-DNA and genomic DNA from the mag1-1 mutant. Primers specific to the T-DNA border, pBI-TR-1 (5′-CGTCAGTGGAGCATTTTTGACAAG-3′) and pBI-TR-2 (5′-TTTGCTAGCTGATAGTGACCTTAG-3′), were used for the first and second amplifications, respectively. A degenerate primer, TAIL-1 [(ATGC)GTCGA(GC)(AT)GA(ATGC)A(AT)GAA], was used. For the nucleotide sequencing of the resulting PCR products, pBI-TR-3 (5′-AAACTCCAGAAACCCGCGGCTGAG-3′) was used. Transformation of the mag1-1 mutant with the MAG1 gene A 4,317 bp Spe I/ Xho I fragment containing the MAG1 gene was cut from bacterial artificial chromosome (BAC) clone T23J7 and subcloned into the binary vector pGreenII0229 (Hellens et al. 2000 ). After sequence verifications, the constructs were introduced into mag1-1 mutant plants via Agrobacterium tumefaciens -mediated transformation using the floral dip method. Transformed plants were subsequently selected on MS medium containing bialaphos (5 mg l −1 ). Preparation of antibodies A full-length cDNA for VPS29 was synthesized with total RNA from rosette leaves and subcloned into pDONR221 (Invtrogen Japan, Tokyo, Japan). Using a GATEWAY system, a DNA fragment that encodes VPS29 was inserted into a pET32-derived vector (Novagen, Madison, WI, USA), in which the recombination site is located at the Eco RV site. A full-length cDNA for VPS35 (U50265) was obtained from the Arabidopsis Biological Resource Center. A Sal I/ Bam HI fragment that contained about seven-eighths the length of the coding region was subcloned into pET28. Recombinant VPS29 and VPS35 proteins were expressed in Escherichia coli , purified on Ni 2+ -chelating columns (Amersham Bioscience, Tokyo, Japan) and used as antigens in the preparation of specific antibodies. The antibodies raised against AtVPS29 and AtVPS35 non-specifically recognized major proteins of leaf tissues ( Fig. 7 ). One of the signals on each immunoblot corresponded to the predicted molecular masses of these proteins. Each signal disappeared on the blot of mag1 mutants or the atvps35 mutant. Protein analysis of seeds Protein extracts were prepared from dry seeds or seedlings in SDS–PAGE sample buffer. Samples were subjected to SDS–PAGE followed by either Coomassie blue staining or blotting to Immobilon-P membranes (Nihon Millipore, Tokyo, Japan). The membranes were treated with antibodies against Arabidopsis 2S albumin (1 : 10,000), Arabidopsis 12S globulin α-subunit (1 : 10,000), AtVPS29 (1 : 5,000) and AtVPS35 (1 : 5,000). Antibodies against Arabidopsis 2S albumin and 12S globulin α-subunit were described previously (Shimada et al. 2003b ). Immunoreactive signals were detected with the ECL detection system (Amersham, Japan). The 49 and 54 kDa proteins that were separated on the blots with Coomassie blue staining were subjected to automatic Edman degradation on a gas-phase sequence analyzer (model 477; Applied Biosystems, Foster City, CA, USA). Total seed proteins were measured with a protein assay kit (Advanced Protein Assay Reagent, Cytoskeleton, Denver, CO, USA) Microscopic analysis For immunoelectron microscopy, we fixed, dehydrated and embedded the dry seeds in LR White resin (London Resin Co., Basingstoke, UK) as described previously (Hayashi et al. 1998 ), except for using the fixative containing 10% dimethylsulfoxide. Samples were treated with antibodies against Arabidopsis 2S albumin (dilution 1 : 50) and Arabidopsis 12S globulin (dilution 1 : 50). Sections were examined with a transmission electron microscope (1200EX; JEOL, Tokyo, Japan) at 80 kV. For laser-scanning confocal microscopy, dry seeds from wild-type and the mag1-1 seeds were pressed in glycerol between the slide glass and coverstrip to push out cotyledons. They were inspected with a fluorescence microscope (Axioplan 2, Carl Zeiss, Jena, Germany) equipped with a confocal laser-scanning unit (CSU10, Yokogawa Electronic Corp., Tokyo, Japan) and a laser unit (Sapphire 488, Coherent, CA, USA). We used the filter set for green fluorescent protein (GFP) fluorescence (510AF23 Omega Optical, VT, USA). Auto-fluorescent images of PSVs were acquired by a CCD camera (OrcaER, Hamamatsu Photonics, Hamamatsu, Japan) and processed by IPLab software (Scanalytics, Fairfax, VA, USA) and Adobe Photoshop 5.5 (Adobe Systems, Tokyo, Japan). RT–PCR and realtime PCR Total RNA was isolated using an RNeasy plant mini kit (QIAGEN, Valencia, CA, USA). A 1 μg aliquot of total RNA was treated by DNase I (Invitrogen, Carlsbad, CA, USA) to reduce the contaminated genomic DNA. Reverse transcription was performed using Ready-To-Go™ RT–PCR beads (Amersham Biosciences) with an oligo(dT) 12−18 primer or random primers. PCR conditions were as follows: 94°C for 2 min; 30 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1.5 min. The following primers were used for PCR: for the MAG1 gene, 5′-ATGGTGCTGGTATTGGCATTGGG-3′ (F1) and 5′-CTACGGACCAGAGCTGGTAGTAGGG-3′ (F2); for the Actin2 gene, 5′-AGAGATTCAGATGCCCAGAAGTCTTGTTCC-3′ and 5′-AACGATTCCTGGACCTGCCTCATCATACTC-3′ (Ratcliffe et al. 2003 ). Real-time PCR was performed using the 7500 Real-Time PCR System (Applied Biosystems Japan, Tokyo, Japan) with a TaqMan probe according to the method recommended by the supplier. PCR was performed in 20 μl volumes using the TaqMan Core PCR Reagent Kit (Applied Biosystems Japan). The sequences of the primers and the probe for the MAG1 gene are 5′-CGCGATACCCTGAGAATAAAACCTT-3′, 5′-CCTGGTGGCCATGACACAAT-3′ and 5′-GCAATTCAAGCTGGG-3′. The probe was labeled with 6-carboxy-fluoroscein (FAM) at its 3′ end. TaqMan Ribosomal RNA Control Reagents were used as a control to detect 18S rRNA. Acknowledgments mag1-2 was generated in the context of the GABI-Kat program and was provided by Bernd Weisshaar (MPI for Plant Breeding Research; Cologne, Germany). This work was supported by CREST of the Japan Science and Technology Corporation, and Grants-in-Aid for Scientific Research (Nos. 16044224, 16085203 and 17107002) and 21st Century COE Research Kyoto University (A14) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Arighi CN,  Hartnell LM,  Aguilar RC,  Haft CR,  Bonifacino JS.  Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor,  J. Cell Biol ,  2004, vol.  165 (pg.  123- 133) Google Scholar CrossRef Search ADS PubMed  Bachhawat AK,  Suhan J,  Jones EW.  The yeast homolog of Hβ58, a mouse gene essential for embryogenesis, performs a role in the delivery of proteins to the vacuole,  Genes Dev ,  1994, vol.  8 (pg.  1379- 1387) Google Scholar CrossRef Search ADS PubMed  Bowers K,  Stevens TH.  Protein transport from the late Golgi to the vacuole in the yeast,  Saccharomyces cerevisiae. Biochim. Biophys. Acta ,  2005, vol.  1744 (pg.  438- 454) Google Scholar CrossRef Search ADS   Collins BM,  Skinner CF,  Watson PJ,  Seaman MN,  Owen DJ.  Vps29 has a phosphoesterase fold that acts as a protein interaction scaffold for retromer assembly,  Nat. Struct. Mol. Biol ,  2005, vol.  12 (pg.  594- 602) Google Scholar CrossRef Search ADS PubMed  daSilva LL,  Taylor JP,  Hadlington JL,  Hanton SL,  Snowden CJ,  Fox SJ,  Foresti O,  Brandizzi F,  Denecke J.  Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting,  Plant Cell ,  2005, vol.  17 (pg.  132- 148) Google Scholar CrossRef Search ADS PubMed  Haft CR,  de la Luz Sierra M,  Bafford R,  Lesniak MA,  Barr VA,  Taylor SI.  Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes,  Mol. Biol. Cell ,  2000, vol.  11 (pg.  4105- 4116) Google Scholar CrossRef Search ADS PubMed  Hara-Nishimura I,  Matsushima R,  Shimada T,  Nishimura M.  Diversity and formation of endoplasmic reticulum-derived compartments in plants. Are these compartments specific to plant cells?,  Plant Physiol ,  2004, vol.  136 (pg.  3435- 3439) Google Scholar CrossRef Search ADS PubMed  Hara-Nishimura I,  Shimada T,  Hatano K,  Takeuchi Y,  Nishimura M.  Transport of storage proteins to protein-storage vacuoles is mediated by large precursor-accumulating vesicles,  Plant Cell ,  1998, vol.  10 (pg.  825- 836) Google Scholar CrossRef Search ADS PubMed  Hayashi M,  Toriyama K,  Kondo M,  Nishimura M.  2,4-Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid beta-oxidation,  Plant Cell ,  1998, vol.  10 (pg.  183- 195) Google Scholar PubMed  Hellens RP,  Edwards EA,  Leyland NR,  Bean S,  Mullineaux PM.  pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation,  Plant Mol. Biol ,  2000, vol.  42 (pg.  819- 832) Google Scholar CrossRef Search ADS PubMed  Hillmer S,  Movafeghi A,  Robinson DG,  Hinz G.  Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus,  J. Cell Biol ,  2001, vol.  152 (pg.  41- 50) Google Scholar CrossRef Search ADS PubMed  Horazdovsky BF,  Davies BA,  Seaman MN,  McLaughlin SA,  Yoon S,  Emr SD.  A sorting nexin-1 homologue, Vps5p, forms a complex with Vps17p and is required for recycling the vacuolar protein-sorting receptor,  Mol. Biol. Cell ,  1997, vol.  8 (pg.  1529- 1541) Google Scholar CrossRef Search ADS PubMed  Jiang L,  Phillips TE,  Rogers SW,  Rogers JC.  Biogenesis of the protein storage vacuole crystalloid,  J. Cell Biol ,  2000, vol.  150 (pg.  755- 770) Google Scholar CrossRef Search ADS PubMed  Jolliffe NA,  Brown JC,  Neumann U,  Vicre M,  Bachi A,  Hawes C,  Ceriotti A,  Roberts LM,  Frigerio L.  Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors ,  Plant J ,  2004, vol.  39 (pg.  821- 833) Google Scholar CrossRef Search ADS PubMed  Jolliffe NA,  Craddock CP,  Frigerio L.  Pathways for protein transport to seed storage vacuoles,  Biochem. Soc. Trans ,  2005, vol.  33 (pg.  1016- 1018) Google Scholar CrossRef Search ADS PubMed  Liu YG,  Mitsukawa N,  Oosumi T,  Whittier RF.  Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR ,  Plant J ,  1995, vol.  8 (pg.  457- 463) Google Scholar CrossRef Search ADS PubMed  Marcusson EG,  Horazdovsky BF,  Cereghino JL,  Gharakhanian E,  Emr SD.  The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene,  Cell ,  1994, vol.  77 (pg.  579- 586) Google Scholar CrossRef Search ADS PubMed  Masclaux FG,  Galaud JP,  Pont-Lezica R.  The riddle of the plant vacuolar sorting receptors,  Protoplasma ,  2005, vol.  226 (pg.  103- 108) Google Scholar CrossRef Search ADS PubMed  Oliviusson P,  Heinzerling O,  Hillmer S,  Hinz G,  Tse YC,  Jiang L,  Robinson DG.  Plant retromer, localized to the prevacuolar compartment and microvesicles in Arabidopsis, may interact with vacuolar sorting receptors,  Plant Cell ,  2006, vol.  18 (pg.  1239- 1252) Google Scholar CrossRef Search ADS PubMed  Paravicini G,  Horazdovsky BF,  Emr SD.  Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases,  Mol. Biol. Cell ,  1992, vol.  3 (pg.  415- 427) Google Scholar CrossRef Search ADS PubMed  Park M,  Lee D,  Lee GJ,  Hwang I.  AtRMR1 functions as a cargo receptor for protein trafficking to the protein storage vacuole,  J. Cell Biol ,  2005, vol.  170 (pg.  757- 767) Google Scholar CrossRef Search ADS PubMed  Ratcliffe OJ,  Kumimoto RW,  Wong BJ,  Riechmann JL.  Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold,  Plant Cell ,  2003, vol.  15 (pg.  1159- 1169) Google Scholar CrossRef Search ADS PubMed  Robinson DG,  Oliviusson P,  Hinz G.  Protein sorting to the storage vacuoles of plants: a critical appraisal,  Traffic ,  2005, vol.  6 (pg.  615- 625) Google Scholar CrossRef Search ADS PubMed  Schmid M,  Davison TS,  Henz SR,  Pape UJ,  Demar M,  Vingron M,  Scholkopf B,  Weigel D,  Lohmann JU.  A gene expression map of Arabidopsis thaliana development ,  Nat. Genet ,  2005, vol.  37 (pg.  501- 506) Google Scholar CrossRef Search ADS PubMed  Seaman MN,  Marcusson EG,  Cereghino JL,  Emr SD.  Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products,  J. Cell Biol ,  1997, vol.  137 (pg.  79- 92) Google Scholar CrossRef Search ADS PubMed  Seaman MN,  McCaffery JM,  Emr SD.  A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast,  J. Cell Biol ,  1998, vol.  142 (pg.  665- 681) Google Scholar CrossRef Search ADS PubMed  Shimada T,  Fuji K,  Tamura K,  Kondo M,  Nishimura M,  Hara-Nishimura I.  Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana,  Proc. Natl Acad. Sci. USA ,  2003a, vol.  100 (pg.  16095- 16100) Google Scholar CrossRef Search ADS   Shimada T,  Kuroyanagi M,  Nishimura M,  Hara-Nishimura I.  A pumpkin 72-kDa membrane protein of precursor accumulating vesicles has characteristics of a vacuolar sorting receptor,  Plant Cell Physiol ,  1997, vol.  38 (pg.  1414- 1420) Google Scholar CrossRef Search ADS PubMed  Shimada T,  Watanabe E,  Tamura K,  Hayashi Y,  Nishimura M,  Hara-Nishimura I.  A vacuolar sorting receptor PV72 on the membrane of vesicles that accumulate precursors of seed storage proteins (PAC vesicles),  Plant Cell Physiol ,  2002, vol.  43 (pg.  1086- 1095) Google Scholar CrossRef Search ADS PubMed  Shimada T,  Yamada K,  Kataoka M,  Nakaune S,  Koumoto Y, et al.  Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana,  J. Biol. Chem ,  2003b, vol.  278 (pg.  32292- 32299) Google Scholar CrossRef Search ADS   Wang D,  Guo M,  Liang Z,  Fan J,  Zhu Z,  Zang J,  Li X,  Teng M,  Niu L,  Dong Y,  Liu P.  Crystal structure of human vacuolar protein sorting protein 29 reveals a phosphodiesterase/nuclease-like fold and two protein–protein interaction sites,  J. Biol. Chem ,  2005, vol.  280 (pg.  22962- 22967) Google Scholar CrossRef Search ADS PubMed  Watanabe E,  Shimada T,  Kuroyanagi M,  Nishimura M,  Hara-Nishimura I.  Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin,  J. Biol. Chem ,  2002, vol.  277 (pg.  8708- 8715) Google Scholar CrossRef Search ADS PubMed  Abbreviations: Abbreviations: CPY carboxypeptidase Y ER endoplasmic reticulum Mag Maigo PAC vesicle precursor-accumulating vesicle PSV protein storage vacuole PVC pre-vacuolar compartment RT–PCR reverse transcription–PCR TAIL-PCR thermal asymmetric interlaced PCR VPS vacuolar protein sorting VSR vacuolar sorting receptor. © The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
TI - AtVPS29, a Putative Component of a Retromer Complex, is Required for the Efficient Sorting of Seed Storage Proteins
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcj103
DA - 2006-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/atvps29-a-putative-component-of-a-retromer-complex-is-required-for-the-B0RvqBVTfe
SP - 1187
EP - 1194
VL - 47
IS - 9
DP - DeepDyve
ER -