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Selective Effects of Calcium Chelators on Anterograde and Retrograde Protein Transport in the Cell

Selective Effects of Calcium Chelators on Anterograde and Retrograde Protein Transport in the Cell THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 38, Issue of September 20, pp. 35682–35687, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Selective Effects of Calcium Chelators on Anterograde and Retrograde Protein Transport in the Cell* Received for publication, April 29, 2002, and in revised form, July 8, 2002 Published, JBC Papers in Press, July 11, 2002, DOI 10.1074/jbc.M204157200 Ji-Long Chen‡, Jatinder P. Ahluwalia§, and Mark Stamnes‡¶ From the Departments of ‡Physiology & Biophysics and §Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242 Calcium plays a regulatory role in several aspects of Both major organelles of the early secretory pathway, the protein trafficking in the cell. Both vesicle fusion and endoplasmic reticulum and the Golgi apparatus, are known to vesicle formation can be inhibited by the addition of be cellular calcium stores (6, 7). Secretory vesicles have also calcium chelators. Because the effects of calcium chela- been shown recently to act as a dynamic calcium store (8). Two tors have been studied predominantly in cell-free sys- classes of ATP-dependent pump proteins maintain high lume- tems, it is not clear exactly which transport steps in the nal calcium concentrations in these organelles. The sarco/en- secretory pathway are sensitive to calcium levels. In this doplasmic reticulum calcium ATPase (SERCA)-type pumps are regard, we have studied the effects of calcium chelators found both on the ER and the Golgi (9 –11). Pmr1p was iden- on both anterograde and retrograde protein transport tified as a Golgi calcium pump in yeast (12), and mutations in in whole cells. Using both cytochemical and biochemical the mammalian isoform lead to Hailey-Hailey disease (13). analyses, we find that the anterograde-directed exit of Studies analyzing the subcellular localization of calcium indi- vesicular stomatitis virus G protein and the retrograde- cate that calcium concentrations are particularly high in the directed exit of Shiga toxin from the Golgi apparatus are Golgi region of cells and that calcium gradients could exist both inhibited by calcium chelation. The exit of vesicu- among organelles of the secretory pathway (6, 14). Both the ER lar stomatitis virus G from a pre-Golgi compartment and and the Golgi apparatus have also been shown to have inositol the exit of Shiga toxin from an endosomal compartment are sensitive to the membrane-permeant calcium chela- 1,4,5-trisphosphate receptors for the triggered release of cal- tor 1,2-bis(2-amino phenoxy)ethane-N,N,N,N-tetraace- cium during cell signaling (1, 15). Specific calcium binding or tic acid–tetrakis (acetoxymethyl ester) (BAPTA-AM). By sequestering proteins have been localized to each organelle. contrast, endoplasmic reticulum exit and endocytic in- The calcium-binding chaperone proteins calreticulin and cal- ternalization from the plasma membrane are not af- nexin function in the ER (16, 17). At least three lumenal cal- fected by BAPTA. Together, our data show that some, cium-binding proteins, Cab45, CALNUC (nucleobindin), and but not all, trafficking steps in the cell may be regulated p54/NEFA have been localized to the Golgi (11, 18, 19). The by calcium. These studies provide a framework for a presence of calcium stores and specific calcium-binding pro- more detailed analysis of the role of calcium as a regu- teins in secretory organelles has revived the notion that cal- latory agent during protein transport. cium or calcium gradients are important for regulating the constitutive secretory pathway (20). Studies on ER to Golgi transport in yeast and in semi-intact The modulation of cytosolic calcium levels serves as an (perforated) cells have indeed indicated a role for calcium in important signaling system for cell regulation (1). Protein this trafficking step (21–24). Regulation by calcium has also transport within the secretory pathway is among the processes been implicated in homotypic vacuolar fusion (25), in late en- regulated by calcium. The best characterized role for calcium in dosome-lysosome heterotypic fusion and the reformation of ly- vesicular transport is in the calcium-triggered fusion of synap- sosomes from hybrid organelles (26), and in fusion between tic and secretory vesicles at the plasma membrane (2– 4). endosomal compartments (27). There are several lines of evi- Calcium-regulated vesicle fusion and fusion reactions in the dence showing that calcium is important for the function of the constitutive secretory pathway have similar molecular mecha- Golgi apparatus. Most directly, calcium chelators have been nisms. For example, both types of fusion reactions are medi- 1 shown to block intra-Golgi protein transport in vitro (28). The ated or regulated by SNARE proteins, as well as rab- and brefeldin A-induced retrograde transport from the Golgi appa- sec1/munc18-related proteins (5). Recent studies indicate that ratus to the endoplasmic reticulum is also affected by calcium a regulatory role for calcium may also extend to fusion reac- chelation (29). Calmodulin has been shown to mediate the tions in the constitutive secretory pathway. effects of calcium on several of these fusion or transport reac- tions (25, 28, 30). Interestingly, the calcium chelator BAPTA is * This work was supported by grants (to M. S.) from the American often found to be more effective than EGTA at inhibiting these Cancer Society, The American Heart Association Heartland Affiliate, membrane fusion reactions (25–27). BAPTA has a much faster and the Roy J. Carver Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This on rate for calcium binding than does EGTA. This observation article must therefore be hereby marked “advertisement” in accordance is frequently interpreted to indicate that calcium transients or with 18 U.S.C. Section 1734 solely to indicate this fact. gradients rather than steady-state calcium levels regulate To whom correspondence should be addressed. E-mail: mark- these processes. [email protected]. The abbreviations used are: SNARE, soluble N-ethylmaleimide- We have recently reported that in addition to regulating sensitive factor attachment protein receptors; ER, endoplasmic reticu- membrane fusion reactions, calcium may play a role in regu- lum; BAPTA-AM, 1,2-bis(2-amino phenoxy)ethane-N,N,N,N-tetraace- lating the coating or uncoating of transport vesicles. We found tic acid–tetrakis (acetoxymethyl ester); VSV, vesicular stomatitis virus; that treating cells with the membrane-permeable calcium PBS, phosphate-buffered saline; STB, Shiga toxin B-fragment; NRK, normal rat kidney; endo H, endoglycosidase H. chelator BAPTA-AM led to the dissociation of the COPI vesicle 35682 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Calcium Regulation of Protein Transport 35683 umn (Bio-Rad) was used in place of Mono Q column. The STB was coat protein, coatomer, from the Golgi apparatus (31). Using labeled using activated Cy2 for fluorescence microscopy (Amersham cell-free Golgi binding assays, we also observed that the addi- Biosciences; HP7 9NA) or activated biotin for Western blot analysis tion of BAPTA led to the rapid dissociation of coatomer from according to the manufacturer’s instructions. The cells were incubated previously coated Golgi membranes. As with fusion, BAPTA g/ml labeled STB. The cells were then for 30 min in the presence of 2 was more effective than EGTA at mediating this effect. The washed three times with fresh medium and incubated for various times addition of calcium chelators appears to affect a step in vesicle as described in the figure legends. Western Blotting and Glycosylation Analysis—The proteins were coat assembly after the recruitment of ADP-ribosylation factor, fractionated using SDS-PAGE and blotted onto polyvinylidene difluo- the small GTP-binding protein that triggers coat assembly. ride membranes using standard protocol for the Bio-Rad minigel and The previous studies strongly implicate a role for calcium in blotting apparatuses. Following the transfer, the membranes were in- regulating transport through the constitutive exocytic and en- cubated with appropriate dilutions of the indicated primary antibodies. docytic protein transport pathways. These studies have utilized The signal was visualized using horseradish peroxidase-conjugated sec- cell-free systems, and thus it is still not clear to what extent ondary antibodies (Bio-Rad) and ECL (Amersham Biosciences). The treatment of VSV G protein with endoglycosidase H (Calbio- calcium modulates transport through the secretory system in chem) was done according to the manufacturer’s instructions. For STB whole cells. As a step toward clarifying the role of calcium in glycosylation analysis, the toxin was labeled with biotin (Pierce) using transport, we have analyzed the effects of membrane-permeant appropriate molar ratio of biotin to protein. STB-biotin was detected by calcium chelators on both anterograde and retrograde protein Western blotting using anti-biotin antibodies. In some experiments, the transport through the secretory pathway. This study indicates cells were incubated in the presence of 1 g/ml tunicamycin 1 h before that select transport steps are sensitive to calcium transients the addition of STB. or gradients. RESULTS EXPERIMENTAL PROCEDURES BAPTA Blocks Transport from an ER/Golgi Intermediate Materials—Tissue culture media were obtained through the Diabe- Compartment to the Golgi but Does Not Affect Exit from the tes and Endocrinology Research Center, University of Iowa. The con- ER—We used a NRK cell line constitutively expressing a tem- struct pT77-SLT-B-Glyc-KDEL was obtained as gifts from Drs. B. Goud perature-sensitive VSV G (ts045) to analyze the effects of cal- and A. Girod. BAPTA-AM and EGTA-AM were purchased from Molec- ular Probes (Eugene, OR). The following antibodies were used in this cium chelation on anterograde protein transport through the study: anti-rat mannosidase II (Berkeley Antibody Co., Richmond, CA), secretory pathway. VSV G (ts045) is trapped in the ER at the polyclonal anti-VSV G (Medical & Biological Laboratories Co., Naka-Ku restrictive temperature (39.5 °C) but is transported normally Nagoya, Japan), P5D4 monoclonal anti-VSV G (32), anti-BiP (Affinity through the Golgi apparatus to the plasma membrane at the Bioreagents Inc., Golden, CO), and anti-biotin (Sigma). permissive temperature (32 °C). The transport of VSV G was VSV G Protein Transport Assay—The cell line Gts-NRK (a gift from monitored using both fluorescence microscopy (Fig. 1) and by Dr. V. Malhotra) that stably expresses the temperature-sensitive ts045- VSV G was maintained by growth at a permissive temperature (32 °C) determining the glycosylation state of the VSV G (Fig. 2). in -minimum essential medium plus 5% fetal calf serum. The cells Following a 16-h incubation at the restrictive temperature, the were incubated for 14 –16 h at a restrictive temperature (39.5 °C) to VSV G is localized to a dispersed ER compartment (Fig. 1A). accumulate VSV G protein in the ER and processed for immunofluo- When cells were switched to a permissive temperature (32 °C) rescence. For VSV G protein transport assay, the cells were incubated for 15 min, the VSV G was relocated to the juxtanuclear Golgi at 39.5 °C for 14 –16 h, and then VSV G protein was released from the compartment (Fig. 1B). After 90 min at the permissive temper- temperature block by switching the cells to 32 °C medium containing 10 g/ml cycloheximide. The cells were incubated for 15 or 90 min at ature, VSV G was found dispersed in small punctate and tu- (32 °C) and processed for immunofluorescence. BAPTA-AM or bular structures and at the cell surface (Fig. 1C). In some cells, EGTA-AM was added at the indicated times by replacing the medium a small amount of residual Golgi-like staining was also ob- with medium containing 50 M BAPTA-AM or 50 M EGTA-AM plus 10 served. ER-localized, core-glycosylated VSV G protein is sensi- g/ml cycloheximide. Where indicated, 2 M A23187 (Sigma), 3 mM tive to deglycosylation by endoglycosidase H (endo H), whereas EGTA, or 5 mM CaCl were also added to the medium. complex oligosaccharides generated by processing in the Golgi Immunofluorescence—Gts-NRK cells were plated onto glass cover slips, and after 24 h the cells were washed with phosphate-buffered are endo H-resistant. Endo H digestion of cell extracts follow- saline (PBS), fixed with 4% paraformaldehyde, and permeabilized using ing the incubation at the restrictive temperature confirms that 0.1% Triton X-100 for 4 min at room temperature. The formaldehyde the VSV G from these cells is completely endo H-sensitive and was quenched with 50 mM ammonium chloride for 10 min at room thus has not been processed by the Golgi (Fig. 2A, lanes 1 and temperature. The cells were washed three times with PBS and blocked 2). Following a 90-min incubation at the permissive tempera- with 2.5% donkey serum in PBS for1hat room temperature. Appro- ture, all of the VSV G in cells is endo H-resistant, indicating priate dilutions of the anti VSV G, anti-mannosidase II, and anti-BiP antibodies in the blocking buffer were added to the cells for1hat room that it has been transported through the Golgi (Fig. 2A, lanes 3 temperature. The cells were washed three times with PBS and incu- and 4). bated with fluorescein isothiocyanate-conjugated anti-rabbit and Texas To determine what effect calcium chelation had on VSV G Red-conjugated anti-mouse secondary antibodies in blocking buffer for transport, we treated cells with the membrane-permeant cal- 1 h at room temperature. The cells were washed three times, mounted cium chelator BAPTA-AM at various times relative to the on slides, and analyzed on a confocal microscope (Bio-Rad). switch from the restrictive to the permissive temperature. Electron Microscopy—Gts-NRK cells were incubated with or without 50 M BAPTA-AM in -minimum essential medium for 90 min at When BAPTA-AM was added to the cells either 90 min (not 39.5 °C. The cells were fixed in 2.5% glutaraldehyde, post-fixed in 1% shown) or 30 min (Fig. 1D) prior to the switch from the restric- OsO , dehydrated, and embedded in Spurr’s embedding medium. Ul- tive to the permissive temperature, we observed a marked trathin sections stained with uranyl acetate and lead citrate were difference in the localization of VSV G. Instead of localizing to examined with an H-7000 transmission electron microscope. the dispersed post-Golgi structures and the plasma membrane, Shiga Toxin Transport—To construct a vector expressing Shiga toxin VSV G was now found to localize to large, brightly labeled B-fragment (STB) with an N-glycosylation site, PCR was employed. PCR primers T7 (5-TAA TAC GAC TCA CTA TAG GG-3) and STB- perinuclear punctate structures (Fig. 1, compare C and D). WT1 (5-AAT GGA TCC TCA TTC AGA GCT AGT AGA A-3) were used We examined whether the VSVG-containing punctate struc- with plasmid pT77-SLT-B-Glyc-KDEL. The resulting fragment was tures observed upon BAPTA-AM treatment were a pre- or verified by sequencing and then cloned into the pET11a vector (Strat- post-Golgi compartment by testing whether the VSV G was agene, La Jolla, CA) to make pETSTB-Glyc. endo H-resistant under this condition. Fig. 2A shows that when The recombinant Shiga toxin B-fragment was overexpressed in the BAPTA-AM was added at least 30 min prior to switching the BL21(DE3)pLysS bacterial strain (Stratagene) and purified using ion exchange chromatography as described (33) except that a High Q col- cells to the permissive temperature, the majority of the VSV G 35684 Calcium Regulation of Protein Transport FIG.1. Anterograde VSV G trans- port is blocked in the presence of BAPTA. Shown are confocal micrographs of Gts-NRK cells in which the VSV G pro- tein has been immunolabeled. The cells were grown at 39.5 °C for 16 h to accumu- late ts-045 VSV G in the ER (A). The cells were then shifted to the permissive tem- perature (32 °C) for 15 min (B) or for 90 min (C–F). The cells were either un- treated (A–C) or treated with 50 M BAPTA-AM at the times minus 30 min (D), 0 min (E), or plus 30 min (F), relative to the shift to the permissive tempera- ture. The bar indicates 10 M. the ER and then become trapped in a punctate ER/Golgi inter- mediate compartment. We examined the morphology of both the ER and the Golgi following BAPTA treatment to differen- tiate between these possibilities. As shown in Fig. 3, immuno- fluorescence using anti-mannosidase II antibodies to label the Golgi (C and D) and anti-Bip antibodies to label the ER (A and B) revealed that BAPTA treatment had little or no effect on the morphology of either of these organelles. In particular, no punc- tate Bip-positive ER elements analogous to the VSV G-contain- ing structures shown in Fig. 1D were observed. As expected from our previous studies (31), coatomer was found to be dis- sociated from the Golgi after treatment with BAPTA-AM (not shown). Ultrastructural characterization also reveals that the morphology of the Golgi apparatus is not overtly affected in cells following treatment with the calcium chelator (Fig. 3, E and F). The electron micrographs show that the Golgi is of FIG.2. The early addition of BAPTA-AM blocks VSV G trans- port to the Golgi. Shown are Western blots of Gts-NRK cell lysates similar size and retains a stacked structure after treatment probed with anti-VSV G antibodies. A, prior to the lysis the cells had with BAPTA for 90 min. Together, our results reveal that been incubated at the restrictive temperature (39.5 °C) for 16 h (lanes 1 addition of BAPTA-AM and thus a decrease in cytosolic free and 2) or incubated at the restrictive temperature for 16 h and then calcium does not block the exit of VSV G from the ER. However, shifted to the permissive temperature (32 °C) for 90 min (lanes 3–12). As in Fig. 1, BAPTA-AM was omitted (lanes 1– 4) or added (lanes 5–12) it prevents VSV G transport to the Golgi apparatus and causes at the indicated time relative to the temperature switch. Endoglycosi- the accumulation of VSV G in a punctate perinuclear ER/Golgi dase H (EndoH) was added to the samples where indicated to determine intermediate compartment. whether Golgi processing of oligosaccharide chains had occurred. B, The Exit of VSV G from the Golgi Is Blocked by BAPTA—The cells were treated exactly as in A except that A23187 (2 M), CaCl (5 mM), or EGTA (3 mM) was added 30 min prior to the temperature switch addition of BAPTA-AM to the cells simultaneously with the in addition to or instead of the BAPTA-AM as indicated. N/A, not switch to the permissive temperature caused VSV G to accu- applicable. mulate in a juxtanuclear structure reminiscent of the Golgi apparatus (Fig. 1E). Colocalization studies revealed that in- deed, under this condition, VSV G localization overlaps almost was endo H-sensitive and thus had not reached the Golgi (lanes completely with the Golgi marker mannosidase II (Fig. 4). Fig. 5– 8). Fig. 2B shows that the block in ER to Golgi transport by 2A shows that when BAPTA-AM is added at the same time as BAPTA-AM is reversed when intracellular calcium levels are restored by the addition of the calcium ionophore A23187 and the temperature switch, all of the VSV G becomes endo H- resistant (lanes 9 and 10), confirming that it has now reached CaCl to the medium. Together, the fluorescence microscopy and biochemical analysis demonstrate that when BAPTA-AM the Golgi. Together, the data indicate two distinct blocks in anterograde protein transport of VSV G in the presence of is added prior to the switch to the permissive temperature, VSV G is blocked in a pre-Golgi compartment. BAPTA-AM: one block in an ER/Golgi intermediate compart- The punctate perinuclear structures could result from an ment and a second block either within or from the Golgi alteration in ER morphology caused by the presence of BAPTA. apparatus. Alternatively, in the presence of BAPTA, the VSV G might exit We were not able to detect any additional effects of calcium Calcium Regulation of Protein Transport 35685 FIG.5. EGTA-AM is less effective at disrupting anterograde protein transport. Shown are confocal micrographs of Gts-NRK cells labeled with anti VSV G antibodies. The cells were incubated at 39.5 °C and then shifted to 32 °C for 15 min (A and B)or90min(C and D). The cells were either left untreated (A and C) or exposed to 50 M EGTA-AM (B and D) starting at 30 min prior to the temperature switch. The bar indicates 10 M. to BAPTA than to EGTA (25–27, 31). Because BAPTA has a FIG.3. BAPTA treatment does not alter the morphology of the much faster on-rate for calcium binding, this difference has ER or Golgi. Shown are confocal microgaphs (A–D) and electron mi- been interpreted to indicate that a calcium transient or gradi- crographs (E and F) from untreated (A, C, and E) or BAPTA-AM-treated ent may be involved. If the effects of BAPTA-AM in the whole (B, D, and F) NRK cells. Fixed and permeabilized cells were decorated cell analysis reflect the effects of BAPTA in cell-free fusion and with anti-Bip (A and B) to label the ER or anti-mannosidase II to label the Golgi (C and D). E and F show electron micrographs containing budding assays, we predicted that whole cell trafficking might views of stacked Golgi membranes. The bar in D indicates 10 M, and also be more sensitive to BAPTA than to EGTA. Thus, we the bar in F indicates 50 nm. compared the effects of EGTA-AM and BAPTA-AM on VSV G transport. Fig. 5 shows that there was no obvious accumulation of VSV G in the Golgi or in a pre-Golgi compartment upon treatment with EGTA-AM. Furthermore, biochemical analysis revealed that VSV G became endo H-resistant regardless of when EGTA-AM was added relative to the switch to the per- missive temperature (Fig. 6). Although 50 M BAPTA-AM was a more effective inhibitor than 50 M EGTA-AM, 3 mM EGTA, which is not membrane permeant, inhibited VSVG transport to the Golgi when added together with a calcium ionophore (Fig. 2B, lanes 5 and 6). These findings support the model that FIG.4. VSV G localizes to Golgi membranes when BAPTA is BAPTA-AM inhibits protein transport by disrupting intracel- added at the same time as the temperature switch. Shown is a confocal micrograph of Gts-NRK cells labeled for VSV G and anti- lular calcium levels (see “Discussion”). mannosidase II (Mann II). A merged image of the two signals demon- Distinct Steps in Retrograde Protein Transport Are Sensitive strates colocalization. VSV G was accumulated in the ER by incubation to BAPTA—Although the majority of proteins move through at 39.5 °C, and the cells were switched to 32 °C for 90 min. BAPTA-AM the secretory pathway in an anterograde direction, retrograde had been added at the same time as the switch to the permissive temperature. The bar indicates 10 M. protein transport also plays an important role in the function of the secretory pathway. Retrograde transport is necessary for chelation on VSV G transport in post-Golgi transport steps by the retrieval of escaped resident organelle proteins, for the adding BAPTA-AM after the temperature switch. The addition recycling of proteins that function in transport, and for quality of BAPTA-AM 30 min after the temperature switch caused a control and protein degradation. Some bacterial toxins such as small amount of VSV G to accumulate in the Golgi of some cholera toxin and Shiga toxin have proven to be good tools for cells, but there was no obvious accumulation of VSV G in any studying retrograde protein transport through secretory or- additional structure that might represent a transport interme- ganelles (35–38). These toxins are endocytosed into cells and diate between the Golgi and the plasma membrane (Fig. 1, are then transported from endosomes through the Golgi appa- compare C and F). This finding is consistent with a previous ratus to the ER (35, 39, 40). The toxins are transported from study showing that there are no defects in protein transport the lumen of the ER into the cytosol where they then exert their when a calcium ionophore and EGTA are added to cells after a toxic effect (40). 1-h pulse-chase (34). We have used the Shiga-like toxin B subunit to analyze the Both calcium-sensitive membrane fusion reactions and COPI effects of BAPTA-AM on retrograde protein transport. For coat assembly share the property that they are more sensitive these experiments, recombinant STB was purified and co- 35686 Calcium Regulation of Protein Transport FIG.6. EGTA-AM does not block anterograde transit of VSV G FIG.7. Retrograde transport of Shiga toxin B subunit is through the Golgi apparatus. Shown are Western blots of Gts-NRK blocked in the presence of BAPTA-AM. Shown are fluorescence cell lysates probed with anti-VSV G antibodies. Prior to the lysis, the micrographs of Vero cells that were incubated for 30 min at 37 °C with cells had been incubated at the restrictive temperature (39.5 °C) or Cy-2-labeled STB, washed, and then incubated for an additional2hin incubated at the restrictive temperature and shifted to the permissive the absence of the toxin. BAPTA-AM was added at the indicated time temperature (32 °C) as in Fig. 2. EGTA-AM was omitted or added at the relative to the addition of the STB-Cy2. indicated time relative to the temperature switch. Endoglycosidase H (EndoH) was added to the samples where indicated to determine whether Golgi processing of oligosaccharide chains had occurred. valently labeled with Cy2 (see “Experimental Procedures”). Vero cells were incubated with the labeled toxin for 30 min to allow the toxin to internalize. The noninternalized toxin was then washed away with fresh medium, and the cells were allowed to grow for an additional 0.5–16 h. The location of STB in the cell was monitored by fluorescence microscopy (Fig. 7). In addition, we used a recombinant form of Shiga toxin that was engineered to contain an N-glycosylation site (33, 41) to detect the arrival of the STB in the ER biochemically (Fig. 8). Fluo- rescence microscopy shows that the internalized STB first ap- peared in punctate endosomal compartments and then within 2 h became concentrated in the Golgi (Fig. 7). Diffuse ER-like staining was also observed by microscopy after 2-h incubations. FIG.8. BAPTA-AM blocks the arrival of STB in the ER. Shown As a more precise indicator of STB arrival in the ER, we are Western blot analyses of Vero cell lysates following incubations examined its glycosylation state. Consistent with previous with STB-biotin. A, cells were incubated for 30 min with STB-biotin, washed, and incubated for the indicated times in the absence of STB. studies (33), we observed three distinct STB bands on Western The incubations were done in the presence and the absence of tunica- blots following retrograde transport of STB through the secre- mycin to identify the band resulting from core glycosylation in the ER tory pathway (Fig. 8). We confirmed that the uppermost band (glyc-STB). B, cells were incubated for 30 min with STB-biotin followed corresponds to the core-glycosylated form of STB by treating by a 4-h chase in the absence of STB. Either dimethyl sulfoxide (DMSO) cells with tunicamycin to block core glycosylation (Fig. 8A, solvent or BAPTA-AM were added at the indicated times relative to the addition of the STB-biotin. The BAPTA-AM or dimethyl sulfoxide was lanes 6 –10). Fig. 8A shows that STB starts to arrive in the ER present throughout the remainder of the 4-h chase period. at about 2 h after the addition of the toxin (lane 2). We observed a peak of STB glycosylation at about 4 h after addition of the toxin to the cells (Fig. 8A, lane 3). and arrived in the ER (Fig. 8B, lanes 8 and 10). Interestingly, We determined the effects of calcium chelation on the retro- by comparing the localization of STB with its glycosylation grade transport of STB by adding BAPTA-AM to the cells at state, we found that although STB is not glycosylated when different times relative to the addition of the STB. We found BAPTA is added 60 min after the addition of STB to the cells that when BAPTA-AM was added at the same time or prior to (Fig. 8B, lane 6), some of the STB is localized to a juxtanuclear the addition of STB, the toxin was internalized but appeared to Golgi compartment (Fig. 7). We interpret this to indicate that accumulate in punctate endosomal compartments (Fig. 7). Al- some of the STB has arrived at the Golgi by 60 min and that though the Golgi had normal morphology under these condi- BAPTA has inhibited a Golgi to ER retrograde transport step. tions (Fig. 3), we never observed colocalization of STB with the Thus, as was the case for anterograde transport, it appears Golgi when BAPTA-AM was added early (not shown). Deter- that some retrograde transport steps, such as the internaliza- mination of the glycosylation state of STB confirmed that it tion (endocytosis) of STB, are insensitive to calcium chelation, remained unglycosylated and thus did not reach the ER when whereas other steps, such as endosome to Golgi apparatus and BAPTA-AM was added to the cells at the same time as STB Golgi to ER transport, are inhibited in the presence of the (Fig. 8B, lane 2). The results indicate that although internal- calcium chelator BAPTA. ization of STB into an endosomal compartment is not affected DISCUSSION by calcium chelation, its transport from an endosomal compart- ment to the Golgi is blocked by the presence of the calcium Previous studies have shown that both membrane fusion chelator. Previous studies indicate that STB is transported reactions and vesicle assembly reactions are inhibited by the directly from early endosomes to the Golgi, bypassing the late calcium chelator BAPTA. Because these studies have relied on endosomes (42). Our results indicate a requirement for calcium the cell-free reconstitution of these processes, we have set out in this direct trafficking step. to determine whether steps in constitutive anterograde and We found that addition of BAPTA-AM up to 60 min after the retrograde protein transport are affected by calcium chelators addition of STB completely blocked the transport of STB to the in whole cells. The use of the membrane permeant analogs, ER as revealed by its failure to be glycosylated (Fig. 8B, lanes BAPTA-AM and EGTA-AM, is particularly effective for these 2, 4, and 6). When BAPTA-AM was added 90 or 120 min after studies because the analogs do not bind calcium until they have STB, glycosylated STB was observed, indicating that at least entered the cell and the acetoxymethyl ester moiety has been some STB had moved beyond a BAPTA-sensitive transport step hydrolyzed. Because normal extracellular calcium levels are Calcium Regulation of Protein Transport 35687 maintained, the cells remain attached to their substrate, allow- reactions in the secretory pathway should provide additional ing relatively normal cell morphology to be maintained. This insight into this regulation. has allowed the combined immunohistochemical and biochem- Acknowledgment—We thank Dr. Lois Weismann for helpful ical analysis we report in this paper. discussion. 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Selective Effects of Calcium Chelators on Anterograde and Retrograde Protein Transport in the Cell

Chen, Ji-Long; Ahluwalia, Jatinder P.; Stamnes, Mark
Journal of Biological Chemistry – Sep 1, 2002
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 38, Issue of September 20, pp. 35682–35687, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Selective Effects of Calcium Chelators on Anterograde and Retrograde Protein Transport in the Cell* Received for publication, April 29, 2002, and in revised form, July 8, 2002 Published, JBC Papers in Press, July 11, 2002, DOI 10.1074/jbc.M204157200 Ji-Long Chen‡, Jatinder P. Ahluwalia§, and Mark Stamnes‡¶ From the Departments of ‡Physiology & Biophysics and §Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242 Calcium plays a regulatory role in several aspects of Both major organelles of the early secretory pathway, the protein trafficking in the cell. Both vesicle fusion and endoplasmic reticulum and the Golgi apparatus, are known to vesicle formation can be inhibited by the addition of be cellular calcium stores (6, 7). Secretory vesicles have also calcium chelators. Because the effects of calcium chela- been shown recently to act as a dynamic calcium store (8). Two tors have been studied predominantly in cell-free sys- classes of ATP-dependent pump proteins maintain high lume- tems, it is not clear exactly which transport steps in the nal calcium concentrations in these organelles. The sarco/en- secretory pathway are sensitive to calcium levels. In this doplasmic reticulum calcium ATPase (SERCA)-type pumps are regard, we have studied the effects of calcium chelators found both on the ER and the Golgi (9 –11). Pmr1p was iden- on both anterograde and retrograde protein transport tified as a Golgi calcium pump in yeast (12), and mutations in in whole cells. Using both cytochemical and biochemical the mammalian isoform lead to Hailey-Hailey disease (13). analyses, we find that the anterograde-directed exit of Studies analyzing the subcellular localization of calcium indi- vesicular stomatitis virus G protein and the retrograde- cate that calcium concentrations are particularly high in the directed exit of Shiga toxin from the Golgi apparatus are Golgi region of cells and that calcium gradients could exist both inhibited by calcium chelation. The exit of vesicu- among organelles of the secretory pathway (6, 14). Both the ER lar stomatitis virus G from a pre-Golgi compartment and and the Golgi apparatus have also been shown to have inositol the exit of Shiga toxin from an endosomal compartment are sensitive to the membrane-permeant calcium chela- 1,4,5-trisphosphate receptors for the triggered release of cal- tor 1,2-bis(2-amino phenoxy)ethane-N,N,N,N-tetraace- cium during cell signaling (1, 15). Specific calcium binding or tic acid–tetrakis (acetoxymethyl ester) (BAPTA-AM). By sequestering proteins have been localized to each organelle. contrast, endoplasmic reticulum exit and endocytic in- The calcium-binding chaperone proteins calreticulin and cal- ternalization from the plasma membrane are not af- nexin function in the ER (16, 17). At least three lumenal cal- fected by BAPTA. Together, our data show that some, cium-binding proteins, Cab45, CALNUC (nucleobindin), and but not all, trafficking steps in the cell may be regulated p54/NEFA have been localized to the Golgi (11, 18, 19). The by calcium. These studies provide a framework for a presence of calcium stores and specific calcium-binding pro- more detailed analysis of the role of calcium as a regu- teins in secretory organelles has revived the notion that cal- latory agent during protein transport. cium or calcium gradients are important for regulating the constitutive secretory pathway (20). Studies on ER to Golgi transport in yeast and in semi-intact The modulation of cytosolic calcium levels serves as an (perforated) cells have indeed indicated a role for calcium in important signaling system for cell regulation (1). Protein this trafficking step (21–24). Regulation by calcium has also transport within the secretory pathway is among the processes been implicated in homotypic vacuolar fusion (25), in late en- regulated by calcium. The best characterized role for calcium in dosome-lysosome heterotypic fusion and the reformation of ly- vesicular transport is in the calcium-triggered fusion of synap- sosomes from hybrid organelles (26), and in fusion between tic and secretory vesicles at the plasma membrane (2– 4). endosomal compartments (27). There are several lines of evi- Calcium-regulated vesicle fusion and fusion reactions in the dence showing that calcium is important for the function of the constitutive secretory pathway have similar molecular mecha- Golgi apparatus. Most directly, calcium chelators have been nisms. For example, both types of fusion reactions are medi- 1 shown to block intra-Golgi protein transport in vitro (28). The ated or regulated by SNARE proteins, as well as rab- and brefeldin A-induced retrograde transport from the Golgi appa- sec1/munc18-related proteins (5). Recent studies indicate that ratus to the endoplasmic reticulum is also affected by calcium a regulatory role for calcium may also extend to fusion reac- chelation (29). Calmodulin has been shown to mediate the tions in the constitutive secretory pathway. effects of calcium on several of these fusion or transport reac- tions (25, 28, 30). Interestingly, the calcium chelator BAPTA is * This work was supported by grants (to M. S.) from the American often found to be more effective than EGTA at inhibiting these Cancer Society, The American Heart Association Heartland Affiliate, membrane fusion reactions (25–27). BAPTA has a much faster and the Roy J. Carver Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This on rate for calcium binding than does EGTA. This observation article must therefore be hereby marked “advertisement” in accordance is frequently interpreted to indicate that calcium transients or with 18 U.S.C. Section 1734 solely to indicate this fact. gradients rather than steady-state calcium levels regulate To whom correspondence should be addressed. E-mail: mark- these processes. [email protected]. The abbreviations used are: SNARE, soluble N-ethylmaleimide- We have recently reported that in addition to regulating sensitive factor attachment protein receptors; ER, endoplasmic reticu- membrane fusion reactions, calcium may play a role in regu- lum; BAPTA-AM, 1,2-bis(2-amino phenoxy)ethane-N,N,N,N-tetraace- lating the coating or uncoating of transport vesicles. We found tic acid–tetrakis (acetoxymethyl ester); VSV, vesicular stomatitis virus; that treating cells with the membrane-permeable calcium PBS, phosphate-buffered saline; STB, Shiga toxin B-fragment; NRK, normal rat kidney; endo H, endoglycosidase H. chelator BAPTA-AM led to the dissociation of the COPI vesicle 35682 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Calcium Regulation of Protein Transport 35683 umn (Bio-Rad) was used in place of Mono Q column. The STB was coat protein, coatomer, from the Golgi apparatus (31). Using labeled using activated Cy2 for fluorescence microscopy (Amersham cell-free Golgi binding assays, we also observed that the addi- Biosciences; HP7 9NA) or activated biotin for Western blot analysis tion of BAPTA led to the rapid dissociation of coatomer from according to the manufacturer’s instructions. The cells were incubated previously coated Golgi membranes. As with fusion, BAPTA g/ml labeled STB. The cells were then for 30 min in the presence of 2 was more effective than EGTA at mediating this effect. The washed three times with fresh medium and incubated for various times addition of calcium chelators appears to affect a step in vesicle as described in the figure legends. Western Blotting and Glycosylation Analysis—The proteins were coat assembly after the recruitment of ADP-ribosylation factor, fractionated using SDS-PAGE and blotted onto polyvinylidene difluo- the small GTP-binding protein that triggers coat assembly. ride membranes using standard protocol for the Bio-Rad minigel and The previous studies strongly implicate a role for calcium in blotting apparatuses. Following the transfer, the membranes were in- regulating transport through the constitutive exocytic and en- cubated with appropriate dilutions of the indicated primary antibodies. docytic protein transport pathways. These studies have utilized The signal was visualized using horseradish peroxidase-conjugated sec- cell-free systems, and thus it is still not clear to what extent ondary antibodies (Bio-Rad) and ECL (Amersham Biosciences). The treatment of VSV G protein with endoglycosidase H (Calbio- calcium modulates transport through the secretory system in chem) was done according to the manufacturer’s instructions. For STB whole cells. As a step toward clarifying the role of calcium in glycosylation analysis, the toxin was labeled with biotin (Pierce) using transport, we have analyzed the effects of membrane-permeant appropriate molar ratio of biotin to protein. STB-biotin was detected by calcium chelators on both anterograde and retrograde protein Western blotting using anti-biotin antibodies. In some experiments, the transport through the secretory pathway. This study indicates cells were incubated in the presence of 1 g/ml tunicamycin 1 h before that select transport steps are sensitive to calcium transients the addition of STB. or gradients. RESULTS EXPERIMENTAL PROCEDURES BAPTA Blocks Transport from an ER/Golgi Intermediate Materials—Tissue culture media were obtained through the Diabe- Compartment to the Golgi but Does Not Affect Exit from the tes and Endocrinology Research Center, University of Iowa. The con- ER—We used a NRK cell line constitutively expressing a tem- struct pT77-SLT-B-Glyc-KDEL was obtained as gifts from Drs. B. Goud perature-sensitive VSV G (ts045) to analyze the effects of cal- and A. Girod. BAPTA-AM and EGTA-AM were purchased from Molec- ular Probes (Eugene, OR). The following antibodies were used in this cium chelation on anterograde protein transport through the study: anti-rat mannosidase II (Berkeley Antibody Co., Richmond, CA), secretory pathway. VSV G (ts045) is trapped in the ER at the polyclonal anti-VSV G (Medical & Biological Laboratories Co., Naka-Ku restrictive temperature (39.5 °C) but is transported normally Nagoya, Japan), P5D4 monoclonal anti-VSV G (32), anti-BiP (Affinity through the Golgi apparatus to the plasma membrane at the Bioreagents Inc., Golden, CO), and anti-biotin (Sigma). permissive temperature (32 °C). The transport of VSV G was VSV G Protein Transport Assay—The cell line Gts-NRK (a gift from monitored using both fluorescence microscopy (Fig. 1) and by Dr. V. Malhotra) that stably expresses the temperature-sensitive ts045- VSV G was maintained by growth at a permissive temperature (32 °C) determining the glycosylation state of the VSV G (Fig. 2). in -minimum essential medium plus 5% fetal calf serum. The cells Following a 16-h incubation at the restrictive temperature, the were incubated for 14 –16 h at a restrictive temperature (39.5 °C) to VSV G is localized to a dispersed ER compartment (Fig. 1A). accumulate VSV G protein in the ER and processed for immunofluo- When cells were switched to a permissive temperature (32 °C) rescence. For VSV G protein transport assay, the cells were incubated for 15 min, the VSV G was relocated to the juxtanuclear Golgi at 39.5 °C for 14 –16 h, and then VSV G protein was released from the compartment (Fig. 1B). After 90 min at the permissive temper- temperature block by switching the cells to 32 °C medium containing 10 g/ml cycloheximide. The cells were incubated for 15 or 90 min at ature, VSV G was found dispersed in small punctate and tu- (32 °C) and processed for immunofluorescence. BAPTA-AM or bular structures and at the cell surface (Fig. 1C). In some cells, EGTA-AM was added at the indicated times by replacing the medium a small amount of residual Golgi-like staining was also ob- with medium containing 50 M BAPTA-AM or 50 M EGTA-AM plus 10 served. ER-localized, core-glycosylated VSV G protein is sensi- g/ml cycloheximide. Where indicated, 2 M A23187 (Sigma), 3 mM tive to deglycosylation by endoglycosidase H (endo H), whereas EGTA, or 5 mM CaCl were also added to the medium. complex oligosaccharides generated by processing in the Golgi Immunofluorescence—Gts-NRK cells were plated onto glass cover slips, and after 24 h the cells were washed with phosphate-buffered are endo H-resistant. Endo H digestion of cell extracts follow- saline (PBS), fixed with 4% paraformaldehyde, and permeabilized using ing the incubation at the restrictive temperature confirms that 0.1% Triton X-100 for 4 min at room temperature. The formaldehyde the VSV G from these cells is completely endo H-sensitive and was quenched with 50 mM ammonium chloride for 10 min at room thus has not been processed by the Golgi (Fig. 2A, lanes 1 and temperature. The cells were washed three times with PBS and blocked 2). Following a 90-min incubation at the permissive tempera- with 2.5% donkey serum in PBS for1hat room temperature. Appro- ture, all of the VSV G in cells is endo H-resistant, indicating priate dilutions of the anti VSV G, anti-mannosidase II, and anti-BiP antibodies in the blocking buffer were added to the cells for1hat room that it has been transported through the Golgi (Fig. 2A, lanes 3 temperature. The cells were washed three times with PBS and incu- and 4). bated with fluorescein isothiocyanate-conjugated anti-rabbit and Texas To determine what effect calcium chelation had on VSV G Red-conjugated anti-mouse secondary antibodies in blocking buffer for transport, we treated cells with the membrane-permeant cal- 1 h at room temperature. The cells were washed three times, mounted cium chelator BAPTA-AM at various times relative to the on slides, and analyzed on a confocal microscope (Bio-Rad). switch from the restrictive to the permissive temperature. Electron Microscopy—Gts-NRK cells were incubated with or without 50 M BAPTA-AM in -minimum essential medium for 90 min at When BAPTA-AM was added to the cells either 90 min (not 39.5 °C. The cells were fixed in 2.5% glutaraldehyde, post-fixed in 1% shown) or 30 min (Fig. 1D) prior to the switch from the restric- OsO , dehydrated, and embedded in Spurr’s embedding medium. Ul- tive to the permissive temperature, we observed a marked trathin sections stained with uranyl acetate and lead citrate were difference in the localization of VSV G. Instead of localizing to examined with an H-7000 transmission electron microscope. the dispersed post-Golgi structures and the plasma membrane, Shiga Toxin Transport—To construct a vector expressing Shiga toxin VSV G was now found to localize to large, brightly labeled B-fragment (STB) with an N-glycosylation site, PCR was employed. PCR primers T7 (5-TAA TAC GAC TCA CTA TAG GG-3) and STB- perinuclear punctate structures (Fig. 1, compare C and D). WT1 (5-AAT GGA TCC TCA TTC AGA GCT AGT AGA A-3) were used We examined whether the VSVG-containing punctate struc- with plasmid pT77-SLT-B-Glyc-KDEL. The resulting fragment was tures observed upon BAPTA-AM treatment were a pre- or verified by sequencing and then cloned into the pET11a vector (Strat- post-Golgi compartment by testing whether the VSV G was agene, La Jolla, CA) to make pETSTB-Glyc. endo H-resistant under this condition. Fig. 2A shows that when The recombinant Shiga toxin B-fragment was overexpressed in the BAPTA-AM was added at least 30 min prior to switching the BL21(DE3)pLysS bacterial strain (Stratagene) and purified using ion exchange chromatography as described (33) except that a High Q col- cells to the permissive temperature, the majority of the VSV G 35684 Calcium Regulation of Protein Transport FIG.1. Anterograde VSV G trans- port is blocked in the presence of BAPTA. Shown are confocal micrographs of Gts-NRK cells in which the VSV G pro- tein has been immunolabeled. The cells were grown at 39.5 °C for 16 h to accumu- late ts-045 VSV G in the ER (A). The cells were then shifted to the permissive tem- perature (32 °C) for 15 min (B) or for 90 min (C–F). The cells were either un- treated (A–C) or treated with 50 M BAPTA-AM at the times minus 30 min (D), 0 min (E), or plus 30 min (F), relative to the shift to the permissive tempera- ture. The bar indicates 10 M. the ER and then become trapped in a punctate ER/Golgi inter- mediate compartment. We examined the morphology of both the ER and the Golgi following BAPTA treatment to differen- tiate between these possibilities. As shown in Fig. 3, immuno- fluorescence using anti-mannosidase II antibodies to label the Golgi (C and D) and anti-Bip antibodies to label the ER (A and B) revealed that BAPTA treatment had little or no effect on the morphology of either of these organelles. In particular, no punc- tate Bip-positive ER elements analogous to the VSV G-contain- ing structures shown in Fig. 1D were observed. As expected from our previous studies (31), coatomer was found to be dis- sociated from the Golgi after treatment with BAPTA-AM (not shown). Ultrastructural characterization also reveals that the morphology of the Golgi apparatus is not overtly affected in cells following treatment with the calcium chelator (Fig. 3, E and F). The electron micrographs show that the Golgi is of FIG.2. The early addition of BAPTA-AM blocks VSV G trans- port to the Golgi. Shown are Western blots of Gts-NRK cell lysates similar size and retains a stacked structure after treatment probed with anti-VSV G antibodies. A, prior to the lysis the cells had with BAPTA for 90 min. Together, our results reveal that been incubated at the restrictive temperature (39.5 °C) for 16 h (lanes 1 addition of BAPTA-AM and thus a decrease in cytosolic free and 2) or incubated at the restrictive temperature for 16 h and then calcium does not block the exit of VSV G from the ER. However, shifted to the permissive temperature (32 °C) for 90 min (lanes 3–12). As in Fig. 1, BAPTA-AM was omitted (lanes 1– 4) or added (lanes 5–12) it prevents VSV G transport to the Golgi apparatus and causes at the indicated time relative to the temperature switch. Endoglycosi- the accumulation of VSV G in a punctate perinuclear ER/Golgi dase H (EndoH) was added to the samples where indicated to determine intermediate compartment. whether Golgi processing of oligosaccharide chains had occurred. B, The Exit of VSV G from the Golgi Is Blocked by BAPTA—The cells were treated exactly as in A except that A23187 (2 M), CaCl (5 mM), or EGTA (3 mM) was added 30 min prior to the temperature switch addition of BAPTA-AM to the cells simultaneously with the in addition to or instead of the BAPTA-AM as indicated. N/A, not switch to the permissive temperature caused VSV G to accu- applicable. mulate in a juxtanuclear structure reminiscent of the Golgi apparatus (Fig. 1E). Colocalization studies revealed that in- deed, under this condition, VSV G localization overlaps almost was endo H-sensitive and thus had not reached the Golgi (lanes completely with the Golgi marker mannosidase II (Fig. 4). Fig. 5– 8). Fig. 2B shows that the block in ER to Golgi transport by 2A shows that when BAPTA-AM is added at the same time as BAPTA-AM is reversed when intracellular calcium levels are restored by the addition of the calcium ionophore A23187 and the temperature switch, all of the VSV G becomes endo H- resistant (lanes 9 and 10), confirming that it has now reached CaCl to the medium. Together, the fluorescence microscopy and biochemical analysis demonstrate that when BAPTA-AM the Golgi. Together, the data indicate two distinct blocks in anterograde protein transport of VSV G in the presence of is added prior to the switch to the permissive temperature, VSV G is blocked in a pre-Golgi compartment. BAPTA-AM: one block in an ER/Golgi intermediate compart- The punctate perinuclear structures could result from an ment and a second block either within or from the Golgi alteration in ER morphology caused by the presence of BAPTA. apparatus. Alternatively, in the presence of BAPTA, the VSV G might exit We were not able to detect any additional effects of calcium Calcium Regulation of Protein Transport 35685 FIG.5. EGTA-AM is less effective at disrupting anterograde protein transport. Shown are confocal micrographs of Gts-NRK cells labeled with anti VSV G antibodies. The cells were incubated at 39.5 °C and then shifted to 32 °C for 15 min (A and B)or90min(C and D). The cells were either left untreated (A and C) or exposed to 50 M EGTA-AM (B and D) starting at 30 min prior to the temperature switch. The bar indicates 10 M. to BAPTA than to EGTA (25–27, 31). Because BAPTA has a FIG.3. BAPTA treatment does not alter the morphology of the much faster on-rate for calcium binding, this difference has ER or Golgi. Shown are confocal microgaphs (A–D) and electron mi- been interpreted to indicate that a calcium transient or gradi- crographs (E and F) from untreated (A, C, and E) or BAPTA-AM-treated ent may be involved. If the effects of BAPTA-AM in the whole (B, D, and F) NRK cells. Fixed and permeabilized cells were decorated cell analysis reflect the effects of BAPTA in cell-free fusion and with anti-Bip (A and B) to label the ER or anti-mannosidase II to label the Golgi (C and D). E and F show electron micrographs containing budding assays, we predicted that whole cell trafficking might views of stacked Golgi membranes. The bar in D indicates 10 M, and also be more sensitive to BAPTA than to EGTA. Thus, we the bar in F indicates 50 nm. compared the effects of EGTA-AM and BAPTA-AM on VSV G transport. Fig. 5 shows that there was no obvious accumulation of VSV G in the Golgi or in a pre-Golgi compartment upon treatment with EGTA-AM. Furthermore, biochemical analysis revealed that VSV G became endo H-resistant regardless of when EGTA-AM was added relative to the switch to the per- missive temperature (Fig. 6). Although 50 M BAPTA-AM was a more effective inhibitor than 50 M EGTA-AM, 3 mM EGTA, which is not membrane permeant, inhibited VSVG transport to the Golgi when added together with a calcium ionophore (Fig. 2B, lanes 5 and 6). These findings support the model that FIG.4. VSV G localizes to Golgi membranes when BAPTA is BAPTA-AM inhibits protein transport by disrupting intracel- added at the same time as the temperature switch. Shown is a confocal micrograph of Gts-NRK cells labeled for VSV G and anti- lular calcium levels (see “Discussion”). mannosidase II (Mann II). A merged image of the two signals demon- Distinct Steps in Retrograde Protein Transport Are Sensitive strates colocalization. VSV G was accumulated in the ER by incubation to BAPTA—Although the majority of proteins move through at 39.5 °C, and the cells were switched to 32 °C for 90 min. BAPTA-AM the secretory pathway in an anterograde direction, retrograde had been added at the same time as the switch to the permissive temperature. The bar indicates 10 M. protein transport also plays an important role in the function of the secretory pathway. Retrograde transport is necessary for chelation on VSV G transport in post-Golgi transport steps by the retrieval of escaped resident organelle proteins, for the adding BAPTA-AM after the temperature switch. The addition recycling of proteins that function in transport, and for quality of BAPTA-AM 30 min after the temperature switch caused a control and protein degradation. Some bacterial toxins such as small amount of VSV G to accumulate in the Golgi of some cholera toxin and Shiga toxin have proven to be good tools for cells, but there was no obvious accumulation of VSV G in any studying retrograde protein transport through secretory or- additional structure that might represent a transport interme- ganelles (35–38). These toxins are endocytosed into cells and diate between the Golgi and the plasma membrane (Fig. 1, are then transported from endosomes through the Golgi appa- compare C and F). This finding is consistent with a previous ratus to the ER (35, 39, 40). The toxins are transported from study showing that there are no defects in protein transport the lumen of the ER into the cytosol where they then exert their when a calcium ionophore and EGTA are added to cells after a toxic effect (40). 1-h pulse-chase (34). We have used the Shiga-like toxin B subunit to analyze the Both calcium-sensitive membrane fusion reactions and COPI effects of BAPTA-AM on retrograde protein transport. For coat assembly share the property that they are more sensitive these experiments, recombinant STB was purified and co- 35686 Calcium Regulation of Protein Transport FIG.6. EGTA-AM does not block anterograde transit of VSV G FIG.7. Retrograde transport of Shiga toxin B subunit is through the Golgi apparatus. Shown are Western blots of Gts-NRK blocked in the presence of BAPTA-AM. Shown are fluorescence cell lysates probed with anti-VSV G antibodies. Prior to the lysis, the micrographs of Vero cells that were incubated for 30 min at 37 °C with cells had been incubated at the restrictive temperature (39.5 °C) or Cy-2-labeled STB, washed, and then incubated for an additional2hin incubated at the restrictive temperature and shifted to the permissive the absence of the toxin. BAPTA-AM was added at the indicated time temperature (32 °C) as in Fig. 2. EGTA-AM was omitted or added at the relative to the addition of the STB-Cy2. indicated time relative to the temperature switch. Endoglycosidase H (EndoH) was added to the samples where indicated to determine whether Golgi processing of oligosaccharide chains had occurred. valently labeled with Cy2 (see “Experimental Procedures”). Vero cells were incubated with the labeled toxin for 30 min to allow the toxin to internalize. The noninternalized toxin was then washed away with fresh medium, and the cells were allowed to grow for an additional 0.5–16 h. The location of STB in the cell was monitored by fluorescence microscopy (Fig. 7). In addition, we used a recombinant form of Shiga toxin that was engineered to contain an N-glycosylation site (33, 41) to detect the arrival of the STB in the ER biochemically (Fig. 8). Fluo- rescence microscopy shows that the internalized STB first ap- peared in punctate endosomal compartments and then within 2 h became concentrated in the Golgi (Fig. 7). Diffuse ER-like staining was also observed by microscopy after 2-h incubations. FIG.8. BAPTA-AM blocks the arrival of STB in the ER. Shown As a more precise indicator of STB arrival in the ER, we are Western blot analyses of Vero cell lysates following incubations examined its glycosylation state. Consistent with previous with STB-biotin. A, cells were incubated for 30 min with STB-biotin, washed, and incubated for the indicated times in the absence of STB. studies (33), we observed three distinct STB bands on Western The incubations were done in the presence and the absence of tunica- blots following retrograde transport of STB through the secre- mycin to identify the band resulting from core glycosylation in the ER tory pathway (Fig. 8). We confirmed that the uppermost band (glyc-STB). B, cells were incubated for 30 min with STB-biotin followed corresponds to the core-glycosylated form of STB by treating by a 4-h chase in the absence of STB. Either dimethyl sulfoxide (DMSO) cells with tunicamycin to block core glycosylation (Fig. 8A, solvent or BAPTA-AM were added at the indicated times relative to the addition of the STB-biotin. The BAPTA-AM or dimethyl sulfoxide was lanes 6 –10). Fig. 8A shows that STB starts to arrive in the ER present throughout the remainder of the 4-h chase period. at about 2 h after the addition of the toxin (lane 2). We observed a peak of STB glycosylation at about 4 h after addition of the toxin to the cells (Fig. 8A, lane 3). and arrived in the ER (Fig. 8B, lanes 8 and 10). Interestingly, We determined the effects of calcium chelation on the retro- by comparing the localization of STB with its glycosylation grade transport of STB by adding BAPTA-AM to the cells at state, we found that although STB is not glycosylated when different times relative to the addition of the STB. We found BAPTA is added 60 min after the addition of STB to the cells that when BAPTA-AM was added at the same time or prior to (Fig. 8B, lane 6), some of the STB is localized to a juxtanuclear the addition of STB, the toxin was internalized but appeared to Golgi compartment (Fig. 7). We interpret this to indicate that accumulate in punctate endosomal compartments (Fig. 7). Al- some of the STB has arrived at the Golgi by 60 min and that though the Golgi had normal morphology under these condi- BAPTA has inhibited a Golgi to ER retrograde transport step. tions (Fig. 3), we never observed colocalization of STB with the Thus, as was the case for anterograde transport, it appears Golgi when BAPTA-AM was added early (not shown). Deter- that some retrograde transport steps, such as the internaliza- mination of the glycosylation state of STB confirmed that it tion (endocytosis) of STB, are insensitive to calcium chelation, remained unglycosylated and thus did not reach the ER when whereas other steps, such as endosome to Golgi apparatus and BAPTA-AM was added to the cells at the same time as STB Golgi to ER transport, are inhibited in the presence of the (Fig. 8B, lane 2). The results indicate that although internal- calcium chelator BAPTA. ization of STB into an endosomal compartment is not affected DISCUSSION by calcium chelation, its transport from an endosomal compart- ment to the Golgi is blocked by the presence of the calcium Previous studies have shown that both membrane fusion chelator. Previous studies indicate that STB is transported reactions and vesicle assembly reactions are inhibited by the directly from early endosomes to the Golgi, bypassing the late calcium chelator BAPTA. Because these studies have relied on endosomes (42). Our results indicate a requirement for calcium the cell-free reconstitution of these processes, we have set out in this direct trafficking step. to determine whether steps in constitutive anterograde and We found that addition of BAPTA-AM up to 60 min after the retrograde protein transport are affected by calcium chelators addition of STB completely blocked the transport of STB to the in whole cells. The use of the membrane permeant analogs, ER as revealed by its failure to be glycosylated (Fig. 8B, lanes BAPTA-AM and EGTA-AM, is particularly effective for these 2, 4, and 6). When BAPTA-AM was added 90 or 120 min after studies because the analogs do not bind calcium until they have STB, glycosylated STB was observed, indicating that at least entered the cell and the acetoxymethyl ester moiety has been some STB had moved beyond a BAPTA-sensitive transport step hydrolyzed. Because normal extracellular calcium levels are Calcium Regulation of Protein Transport 35687 maintained, the cells remain attached to their substrate, allow- reactions in the secretory pathway should provide additional ing relatively normal cell morphology to be maintained. This insight into this regulation. has allowed the combined immunohistochemical and biochem- Acknowledgment—We thank Dr. Lois Weismann for helpful ical analysis we report in this paper. discussion. 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