The p115-interactive Proteins GM130 and Giantin Participate in Endoplasmic Reticulum-Golgi Traffic
2001; Elsevier BV; Volume: 276; Issue: 4 Linguagem: Inglês
10.1074/jbc.m007957200
ISSN1083-351X
AutoresCecilia Álvarez, Rafael Garcı́a-Mata, Hans‐Peter Hauri, Elizabeth Sztul,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe transport factor p115 is essential for endoplasmic reticulum (ER) to Golgi traffic. P115 interacts with two Golgi proteins, GM130 and giantin, suggesting that they might also participate in ER-Golgi traffic. Here, we show that peptides containing the GM130 or the giantin p115 binding domain and anti-GM130 and anti-giantin antibodies inhibit transport of vesicular stomatitis virus (VSV)-G protein to a mannosidase II-containing Golgi compartment. To determine whether p115, GM130, and giantin act together or sequentially during transport, we compared kinetics of traffic inhibition. Anti-p115, anti-GM130, and anti-giantin antibodies inhibited transport at temporally distinct steps, with the p115-requiring step before the GM130-requiring stage, and both preceding the giantin-requiring stage. Examination of the distribution of the arrested VSV-G protein showed that anti-p115 antibodies inhibited transport at the level of vesicular-tubular clusters, whereas anti-GM130 and anti-giantin antibodies inhibited after the VSV-G protein moved to the Golgi complex. Our results provide the first evidence that GM130 and giantin are required for the delivery of a cargo protein to the mannosidase II-containing Golgi compartment. These data are most consistent with a model where transport from the ER to thecis/medial-Golgi compartments requires the action of p115, GM130, and giantin in a sequential rather than coordinate mechanism. The transport factor p115 is essential for endoplasmic reticulum (ER) to Golgi traffic. P115 interacts with two Golgi proteins, GM130 and giantin, suggesting that they might also participate in ER-Golgi traffic. Here, we show that peptides containing the GM130 or the giantin p115 binding domain and anti-GM130 and anti-giantin antibodies inhibit transport of vesicular stomatitis virus (VSV)-G protein to a mannosidase II-containing Golgi compartment. To determine whether p115, GM130, and giantin act together or sequentially during transport, we compared kinetics of traffic inhibition. Anti-p115, anti-GM130, and anti-giantin antibodies inhibited transport at temporally distinct steps, with the p115-requiring step before the GM130-requiring stage, and both preceding the giantin-requiring stage. Examination of the distribution of the arrested VSV-G protein showed that anti-p115 antibodies inhibited transport at the level of vesicular-tubular clusters, whereas anti-GM130 and anti-giantin antibodies inhibited after the VSV-G protein moved to the Golgi complex. Our results provide the first evidence that GM130 and giantin are required for the delivery of a cargo protein to the mannosidase II-containing Golgi compartment. These data are most consistent with a model where transport from the ER to thecis/medial-Golgi compartments requires the action of p115, GM130, and giantin in a sequential rather than coordinate mechanism. endoplasmic reticulum N-ethylmaleimide-sensitive fusion factor NSF attachment protein receptors vesicular tubular clusters vesicular stomatitis virus glutathioneS-transferase polyacrylamide gel electrophoresis normal rat kidney endoglycosidase H Multiple proteins participate in the sorting and concentration of cargo at the endoplasmic reticulum (ER)1 exit sites, translocation of transport intermediates on microtubules, and the docking/fusion of transport intermediates to their target membranes (1Bannykh S.I. Nishimura N. Balch W.E. Trends Cell Biol. 1998; 8: 21-25Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Proteins that regulate the docking and fusion events include the soluble Ras-related GTPases (Rab proteins) (2Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (425) Google Scholar, 3Plutner H. Cox A.D. Pind S. Khosravi-Far R. Bourne J.R. Schwaninger R. Der C.J. Balch W.E. J. Cell Biol. 1991; 115: 31-43Crossref PubMed Scopus (304) Google Scholar, 4Pind S.N. Nuoffer C. McCaffery J.M. Plutner H. Davidson H.W. Farquhar M.G. Balch W.E. J. Cell Biol. 1994; 125: 239-252Crossref PubMed Scopus (130) Google Scholar, 5Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Cell. 1990; 62: 317-329Abstract Full Text PDF PubMed Scopus (931) Google Scholar, 6Tisdale E.J. Balch W.E. J. Biol. Chem. 1996; 271: 29372-29379Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), NSF (theN-ethylmaleimide-sensitive fusion factor), and soluble NSF attachment proteins (SNAPs), and the transmembrane vesicular- and target-soluble NSF attachment protein receptors (SNAREs) (7Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2670) Google Scholar, 8Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2025) Google Scholar, 9Hay J.C. Scheller R.H. Curr. Opin. Cell Biol. 1997; 9: 505-512Crossref PubMed Scopus (254) Google Scholar, 10Nichols B.J. Pelham H.R. Biochim. Biophys. Acta. 1998; 1404: 9-31Crossref PubMed Scopus (128) Google Scholar). In mammalian cells, transport from the ER to Golgi requires the action of the Rab1 GTPase (2Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (425) Google Scholar) and the SNAREs, rbet1 (11Zhang T. Wong S.H. Tang B.L. Xu Y. Peter F. Subramaniam V.N. Hong W. J. Cell Biol. 1997; 139: 1157-1168Crossref PubMed Scopus (57) Google Scholar), syntaxin5 (12Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar), Sec22b/ERS-24 (13Zhang T. Wong S.H. Tang B.L. Xu Y. Hong W. Mol. Biol. Cell. 1999; 10: 435-453Crossref PubMed Scopus (54) Google Scholar), GOS–28/GS28 (14Subramaniam V.N. Peter F. Philp R. Wong S.H. Hong W. Science. 1996; 272: 1161-1163Crossref PubMed Scopus (124) Google Scholar), and membrin (15Hay J.C. Chao D.S. Kuo C.S. Scheller R.H. Cell. 1997; 89: 149-158Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In addition, the peripherally associated membrane protein p115 is required for ER to Golgi transport at the level of vesicular tubular clusters (VTCs) downstream of the Rab1-requiring step and upstream of the Ca2+-dependent step (16Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (103) Google Scholar). The recruitment of p115 to membranes is mediated by Rab1, and p115 can interact directly with a subset (membrin, rbet 1, and syntaxin5) of the ER-Golgi SNAREs (17Allan B.B. Moyer B.D. Balch W.E. Science. 2000; 289: 444-448Crossref PubMed Scopus (391) Google Scholar). In addition to its VTC association, p115 directly interacts with two Golgi membrane proteins, GM130 and giantin (18Nelson D.S. Alvarez C. Gao Y.S. Garcia-Mata R. Fialkowski E. Sztul E. J. Cell Biol. 1998; 143: 319-331Crossref PubMed Scopus (119) Google Scholar, 19Sonnichsen B. Lowe M. Levine T. Jamsa E. Dirac-Svejstrup B. Warren G. J. Cell Biol. 1998; 140: 1013-1021Crossref PubMed Scopus (254) Google Scholar). GM130 is an extended rod-like protein (∼130 nm) with coiled-coil domains, initially identified as a component of an insoluble Golgi matrix (20Nakamura N. Rabouille C. Watson R. Nilsson T. Hui N. Slusarewicz P. Kreis T.E. Warren G. J. Cell Biol. 1995; 131: 1715-1726Crossref PubMed Scopus (688) Google Scholar). GM130 is anchored by its C-terminal domain to the cytoplasmic face of the membrane through interaction with the myristoylated protein GRASP-65 (the Golgi reassembly stacking protein 65) (21Barr F.A. Puype M. Vandekerckhove J. Warren G. Cell. 1997; 91: 253-262Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 22Barr F.A. Nakamura N. Warren G. EMBO J. 1998; 17: 3258-3268Crossref PubMed Scopus (207) Google Scholar). The p115 binding domain of GM130 lies within its N-terminal 74 amino acids (23Nakamura N. Lowe M. Levine T.P. Rabouille C. Warren G. Cell. 1997; 89: 445-455Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar,24Linstedt A.D. Jesch S.A. Mehta A. Lee T.H. Garcia-Mata R. Nelson D.S. Sztul E. J. Biol. Chem. 2000; 275: 10196-10201Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). At the ultrastructural level, GM130 localizes predominantly to thecis-Golgi (20Nakamura N. Rabouille C. Watson R. Nilsson T. Hui N. Slusarewicz P. Kreis T.E. Warren G. J. Cell Biol. 1995; 131: 1715-1726Crossref PubMed Scopus (688) Google Scholar). This distribution partially overlaps with p115 labeling the cis-Golgi. However, p115 appears more concentrated on structures associated with the cis-most aspect of the cis-cisterna (18Nelson D.S. Alvarez C. Gao Y.S. Garcia-Mata R. Fialkowski E. Sztul E. J. Cell Biol. 1998; 143: 319-331Crossref PubMed Scopus (119) Google Scholar). The other p115 binding partner, giantin, is an integral component of the Golgi membrane that contains a large N-terminal cytoplasmic domain (>350 kDa) and a C-terminal membrane anchor domain (25Linstedt A.D. Hauri H.P. Mol. Biol. Cell. 1993; 4: 679-693Crossref PubMed Scopus (356) Google Scholar, 26Seelig H.P. Schranz P. Schroter H. Wiemann C. Griffiths G. Renz M. Mol. Cell. Biol. 1994; 14: 2564-2576Crossref PubMed Scopus (44) Google Scholar, 27Sohda M. Misumi Y. Fujiwara T. Noshioka M. Ikehara Y. Biochem. Biophys. Res. Commun. 1994; 205: 1399-1408Crossref PubMed Scopus (42) Google Scholar). Giantin is predicted to form a segmented coiled-coil dimer rod of ∼250 nm. The p115 binding domain has been mapped to the N-terminal 70 amino acids of giantin (24Linstedt A.D. Jesch S.A. Mehta A. Lee T.H. Garcia-Mata R. Nelson D.S. Sztul E. J. Biol. Chem. 2000; 275: 10196-10201Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Giantin has been localized to tubular-cisternal Golgi elements that might represent fenestrated connections between cisternal stacks (26Seelig H.P. Schranz P. Schroter H. Wiemann C. Griffiths G. Renz M. Mol. Cell. Biol. 1994; 14: 2564-2576Crossref PubMed Scopus (44) Google Scholar). Such regions of the Golgi have been proposed to be specialized budding domains (28Weidman P. Roth R. Heuser J. Cell. 1993; 75: 123-133Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 29Ladinsky M.S. Mastronarde D.N. McIntosh J.R. Howell K.E. Staehelin L.A. J. Cell Biol. 1999; 144: 1135-1149Crossref PubMed Scopus (519) Google Scholar). Confocal analysis indicates that giantin and p115 do not extensively colocalize in the Golgi region (30Shima D.T. Haldar K. Pepperkok R. Watson R. Warren G. J. Cell Biol. 1997; 137: 1211-1228Crossref PubMed Scopus (196) Google Scholar). The functions of GM130 and giantin have been studied predominantly in a cell-free assay that is based on the observation that Golgi complex disassembles during mitosis and subsequently re-assembles during late telophase (31Lucocq J.M. Berger E.G. Warren G. J. Cell Biol. 1989; 109: 463-474Crossref PubMed Scopus (192) Google Scholar). In this assay, isolated Golgi membranes are treated with mitotic cytosols to generate Golgi fragments that under experimentally controlled conditions (in the presence of NSF, α- and γ-soluble NSF attachment proteins (SNAPs), and p115) re-assemble into cisternal elements and stacks (32Rabouille C. Levine T.P. Peters J.M. Warren G. Cell. 1995; 82: 905-914Abstract Full Text PDF PubMed Scopus (315) Google Scholar). The interaction between p115 and GM130 appears functionally relevant in this assay since the addition of the N-terminal GM130 peptide that binds p115 inhibits cisternal regrowth and cisternal stacking (23Nakamura N. Lowe M. Levine T.P. Rabouille C. Warren G. Cell. 1997; 89: 445-455Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 33Shorter J. Warren G. J. Cell Biol. 1999; 146: 57-70Crossref PubMed Scopus (143) Google Scholar). Likewise, the addition of the p115 binding N-terminal giantin peptide inhibits cisternal regrowth and cisternal stacking (34Lesa G.M. Seemann J. Shorter J. Vandekerckhove J. Warren G. J. Biol. Chem. 2000; 275: 2831-2836Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), suggesting a functional interaction between p115 and giantin. The requirement for both GM130 and giantin function in the assay is further supported by the ability of anti-GM130 or anti-giantin antibodies to block cisternal regrowth and subsequent cisternal stacking (33Shorter J. Warren G. J. Cell Biol. 1999; 146: 57-70Crossref PubMed Scopus (143) Google Scholar). The role of GM130 and giantin in membrane transport has been largely unexplored, but their close relationship with p115 suggests that these proteins might also participate in ER-Golgi traffic. The involvement of GM130 in traffic was suggested by the finding that expression of a GM130 mutant lacking the p115 binding N terminus inhibited the surface delivery of VSV-G protein and resulted in the disappearance of Golgi cisternae with the concurrent accumulation of small vesicles in the Golgi region (35Seemann J. Jokitalo E.J. Warren G. Mol. Biol. Cell. 2000; 11: 635-645Crossref PubMed Scopus (156) Google Scholar). The role of giantin in trafficking has not been previously analyzed. We have examined the role of GM130 and giantin in ER-Golgi traffic by utilizing a VSV-G ts045 based semi-intact cell transport assay we previously used to document p115 requirement at the VTC stage of transport (16Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (103) Google Scholar). Transport was inhibited by peptides corresponding to the p115 binding N-terminal domains of either GM130 or giantin and by anti-GM130 and anti-giantin antibodies. In all cases, the inhibition occurred before VSV-G protein delivery to the mannosidase II-containingmedial/trans-Golgi compartment. Both anti-GM130 and anti-giantin antibodies inhibited transport subsequently to the stage inhibited by anti-p115 antibodies. In agreement, morphological data showed that the anti-GM130 and the anti-giantin antibodies inhibit at the Golgi level at stages downstream from the VTC step inhibited by anti-p115 antibodies. Analysis of the kinetics of inhibition indicates that GM130 and giantin act at temporally different stages, with the GM130-requiring stage preceding the giantin-requiring stage. Our results document the novel requirement for GM130 and giantin in ER-Golgi transport and suggest that GM130 and giantin do not function simultaneously. Rabbit polyclonal antibodies against p115 (36Barroso M. Nelson D.S. Sztul E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 527-531Crossref PubMed Scopus (111) Google Scholar) and against GM130 (18Nelson D.S. Alvarez C. Gao Y.S. Garcia-Mata R. Fialkowski E. Sztul E. J. Cell Biol. 1998; 143: 319-331Crossref PubMed Scopus (119) Google Scholar) were affinity-purified. For affinity purification, lysates of bacteria expressing glutathioneS-transferase (GST)-p115 fusion protein or GM130 were separated by SDS-PAGE and transferred to nitrocellulose. Nitrocellulose strips containing GST-p115 or GM130 were incubated with immune serum in phosphate-buffered saline, 5% fat-free dried milk, 0.1% Tween 20 for 3 h at room temperature. Bound antibodies were eluted with 0.1m glycine, pH 3.0, neutralized with 1/10 volume 1m phosphate buffer, pH 7.4, and dialyzed against 25 mm HEPES-KOH, pH 7.2. The antibodies were concentrated and then used in transport assays. Monoclonal anti-giantin G1/133 antibody has been described previously (25Linstedt A.D. Hauri H.P. Mol. Biol. Cell. 1993; 4: 679-693Crossref PubMed Scopus (356) Google Scholar). Polyclonal antibodies against mannosidase II were kindly provided by Dr. Marilyn Farquhar (University of California at San Diego). Monoclonal antibodies against mannosidase II were from BabCO (Berkeley, CA). Monoclonal antibodies against VSV-G protein (P5D4) were kindly provided by Dr. Kathryn Howell (University of Colorado Health Sciences Center, Denver, CO). Goat anti-rat and anti-mouse antibodies conjugated with fluorescein isothiocyanate or rhodamine were purchased from Jackson ImmunoResearch (West Grove, PA). GM130 fragment encoding amino acids 1 to 270 was generated with the polymerase chain reaction using the primer pair 5′-CCGGATCCGAATGTCGGAAGAAACCAG with 5′-GTTTCTAGACCATGATTTTCACCTCGTC and a template encoding rat GM130 cDNA in a pBluescript-II vector (18Nelson D.S. Alvarez C. Gao Y.S. Garcia-Mata R. Fialkowski E. Sztul E. J. Cell Biol. 1998; 143: 319-331Crossref PubMed Scopus (119) Google Scholar). GST-GM130/1–270 was engineered by inserting the GM130 fragment into theBamHI-SalI restriction sites of the pGEX-5X-1 vector (Amersham Pharmacia Biotech). GST-giantin fragment (GST-GTN/1–261) containing coils I and II has been described previously (24Linstedt A.D. Jesch S.A. Mehta A. Lee T.H. Garcia-Mata R. Nelson D.S. Sztul E. J. Biol. Chem. 2000; 275: 10196-10201Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). GST fusion protein expression and purification was performed according to the manufacturer's (Amersham Pharmacia Biotech) protocol. The ER to Golgi transport assay was performed as described previously (16Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (103) Google Scholar, 37Schwaninger R. Plutner H. Davidson H.W. Pind S. Balch W.E. Methods Enzymol. 1992; 219: 110-124Crossref PubMed Scopus (14) Google Scholar, 38Beckers C.J. Keller D.S. Balch W.E. Cell. 1987; 50: 523-534Abstract Full Text PDF PubMed Scopus (224) Google Scholar). Briefly, NRK cells were grown on 10-cm Petri dishes (80–90% confluent) and infected with the temperature-sensitive strain of the vesicular stomatitis virus, ts045 VSV at 32 °C for 3–4 h (39Bergmann J.E. Methods Cell Biol. 1989; 32: 85-110Crossref PubMed Scopus (89) Google Scholar). The cells were pulse-labeled with Tran35S-label (200 mCi/ml; ICN, Irvine, CA) at the restrictive temperature (42 °C) for 10 min, chased with complete medium for 5 min, and perforated by hypotonic swelling and scraping. Transport reactions were performed in a final total volume of 40 μl in a buffer containing 25 mm HEPES-KOH, pH 7.2, 75 mm potassium acetate, 2.5 mm magnesium acetate, 5 mm EGTA, 1.8 mm CaCl2, 1 mm N-acetylglucosamine, ATP regeneration system (1 mm ATP, 5 mm creatine phosphate, and 0.2 IU of rabbit muscle creatine phosphokinase), 5 μl of rat liver cytosol, and 5 μl of semi-intact cells in 50 mm Hepes-KOH, pH 7.2, 90 mm potassium acetate. Transport was initiated by transferring cells to 32 °C. After 90 min of incubation, cells were pelleted, resuspended in appropriated buffer, and digested with endoglycosidase H (endo-H) as described previously (37Schwaninger R. Plutner H. Davidson H.W. Pind S. Balch W.E. Methods Enzymol. 1992; 219: 110-124Crossref PubMed Scopus (14) Google Scholar). The samples were analyzed on 8% SDS/PAGE and by fluorography. Transport was quantitated using a GS-700 imaging densitometer (Bio-Rad). In some experiments, increasing concentrations of GST fusion peptides were added to a complete transport mixture containing cytosol and incubated on ice for 30 min to allow the peptides to interact with cytosolic p115. The mixtures were then added to the semi-intact cells, and transport was initiated at 32 °C. In some experiments, increasing concentrations of antibodies were added to a complete transport reaction containing semi-intact cells, transport mixture, and cytosol and incubated on ice for 30 min to allow the antibodies to interact with cellular GM130 or giantin. Transport was then initiated at 32 °C. Kinetic staging experiments were performed as described previously (40Davidson H.W. Balch W.E. Methods Enzymol. 1992; 219: 261-267Crossref PubMed Scopus (5) Google Scholar, 41Tisdale E.J. Mol. Biol. Cell. 1999; 10: 1837-1849Crossref PubMed Scopus (67) Google Scholar). To test binding of cytosolic p115 to cell membranes used in the semi-intact cells transport assay, NRK cells were perforated (by hypotonic swelling and scraping as above) and rinsed with 0.5 m KCl to remove endogenous p115. In some experiments, increasing concentrations of GST-GM130/1–270 fusion peptide were added to a complete transport mixture containing cytosol and incubated on ice for 30 min to allow the peptide to interact with cytosolic p115. The mixtures were then added to semi-intact cells, and the cells were incubated for 60 min at 32 °C. Cells were collected by pelleting and rinsed (two times) with transport buffer, and the cell pellet was processed for SDS-PAGE. The amount of membrane-associated p115 was detected by Western blotting and quantified using GS-700 imaging densitometer (Bio-Rad). In some experiments antibodies were added to the semi-intact cells and incubated on ice for 30 min to allow the antibodies to interact with cellular GM130 or giantin. The cells were then supplemented with complete transport mixture and cytosol and incubated for 60 min at 32 °C. Cells were then processed as above to measure membrane-associated p115. NRK cells plated on coverslips were infected with ts045 VSV at 32 °C for 30 min followed by an incubation at 42 °C for 3 h and then shifted to ice and permeabilized with digitonin (20 mg/ml), as described previously (16Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (103) Google Scholar, 42Plutner H. Davidson H.W. Saraste J. Balch W.E. J. Cell Biol. 1992; 119: 1097-1116Crossref PubMed Scopus (177) Google Scholar). Coverslips were incubated at 32 °C for 90 min in transport mixtures supplemented with various IgGs, as indicated under "Results." Transport was terminated by transferring coverslips to ice and fixing the cells in 3% paraformaldehyde, phosphate-buffered saline for 10 min. The coverslips were then processed for double-label immunofluorescence as described previously (16Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (103) Google Scholar). Cell area and the mean fluorescence were measured with the IP Lab Spectrum software (Signal Analytics). The total cell area, the Golgi region area (defined by the presence of a select Golgi marker; see Fig. 6 legend), and the area of colocalization between the VSV-G protein and the Golgi marker were defined manually. These data are presented as a mean of analyses from five representative cells. To test whether the p115-interacting proteins GM130 and giantin participate in membrane traffic, we added recombinant GM130 or giantin peptide constructs to the semi-intact cell transport assay used previously to document p115 requirement during VSV-G protein transport (16Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (103) Google Scholar). The peptide constructs were generated as GST fusion proteins and contained either the N-terminal 270 amino acids of GM130 (GST-GM130/1–270) or the first 261 amino acids of giantin (GST-GTN/1–261) fused to the C terminus of GST (Fig. 1 A). We previously showed that each construct contains a domain sufficient and necessary for p115 binding (24Linstedt A.D. Jesch S.A. Mehta A. Lee T.H. Garcia-Mata R. Nelson D.S. Sztul E. J. Biol. Chem. 2000; 275: 10196-10201Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The fusion constructs and a control GST protein were expressed in bacteria and purified nearly to homogeneity as judged by Coomassie Blue staining (Fig. 1 B). The SDS-PAGE mobilities of the fusion constructs are appropriate for the specific composition of each peptide (GM130/1–270 is predicted to be 29,565 daltons, whereas GTN/1–261 is predicted to be 30,144 daltons). Increasing concentrations of GST-GM130/1–270 or GST-GTN/1–261 were added to cytosol and incubated on ice for 20 min to allow the peptides to bind to cytosolic p115. The mixtures were then used in the semi-intact cell transport assay. Transport was measured by following the processing of VSV-G protein from a core-glycosylated (endo-H-sensitive) ER form to a more mature (endo-H-resistant) form upon its arrival in a Golgi compartment containing mannosidase II. In agreement with previously published results (6Tisdale E.J. Balch W.E. J. Biol. Chem. 1996; 271: 29372-29379Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 14Subramaniam V.N. Peter F. Philp R. Wong S.H. Hong W. Science. 1996; 272: 1161-1163Crossref PubMed Scopus (124) Google Scholar), when transport was measured with untreated cytosol, ∼60% of VSV-G protein was processed to endo-H-resistant form, and this was set as 100% relative transport (Fig. 1 C, lane 2). When transport was analyzed in the absence of ATP (with an ATP-depleting system), more than 90% of VSV-G protein remained endo-H-sensitive, and this was set as 0% processing (lane 1). The addition of 10 μm GST had negligible effect on transport (lane 3). In contrast, addition of increasing amounts of the GST-GM130/1–270 construct led to a dose-dependent inhibition of VSV-G protein transport (lanes 4–6). Adding the GST-GTN/1–261 construct to the transport assay was also inhibitory. At the lowest concentration tested (2 μm,lane 7), the fusion peptide had no effect on transport, but increasing the concentration to 6 or 8 μmled to a significant (>80%) inhibition of VSV-G protein-processing (lanes 8 and 9, respectively). These data suggest that interactions between p115 and GM130 and between p115 and giantin are required for transport and that both GM130 and giantin function in ER-Golgi traffic. However, we have recently shown that the N-terminal domain of GM130 and the N-terminal domain of giantin interact with the same C-terminal acidic domain of p115 and compete for binding (24Linstedt A.D. Jesch S.A. Mehta A. Lee T.H. Garcia-Mata R. Nelson D.S. Sztul E. J. Biol. Chem. 2000; 275: 10196-10201Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Therefore, the addition of either the GST-GM130/1–270 or the GST-GTN/1–261 fusion peptide will prevent the interaction of p115 with both GM130 and giantin. Consequently, inhibition of transport by either peptide may not distinguish between GM130 alone, giantin alone, or both proteins as required for transport. To further characterize the role of GM130 and giantin in transport, anti-GM130, and anti-giantin antibodies were added to the semi-intact cell assay. Increasing amounts of affinity-purified polyclonal anti-GM130 antibodies were added to transport mixtures containing cytosol. The mixtures were added to permeabilized cells, and the cells were incubated on ice for 30 min to allow the antibodies to bind cellular GM130. Cells were then shifted to 32 °C for 90 min. As shown in Fig.2 A, lanes 3–6, the addition of anti-GM130 antibodies inhibited transport of VSV-G protein in a dose-dependent manner. Transport was blocked 70% when 0.48 μg was present in the assay (lane 6). Preincubation of the antibodies with recombinant full-length GM130 immobilized on nitrocellulose strips efficiently neutralized the inhibitory effect (compare lanes 2 and 7). In contrast, the antibodies remained inhibitory when pre-incubated with recombinant full-length p115 immobilized on nitrocellulose strips (data not shown). The addition of monoclonal antibodies directed against giantin also inhibited VSV-G protein transport. As shown in Fig. 2 B,lanes 3–6, the addition of 0.1- 0.8 μg of anti-giantin antibodies inhibited relative transport of VSV-G protein 10–90%. Heat-inactivating the antibodies neutralized their inhibitory effect (compare lanes 2 and 7). Analogous results were obtained with affinity-purified polyclonal anti-giantin antibodies (data not shown). These data indicate that anti-GM130 and anti-giantin antibodies inhibit VSV-G protein transport and suggest that both GM130 and giantin participate in VSV-G protein movement from the ER to a mannosidase II-containing Golgi compartment. We have shown previously that p115 binds to Golgi membranes by interacting with the N termini of GM130 and giantin (18Nelson D.S. Alvarez C. Gao Y.S. Garcia-Mata R. Fialkowski E. Sztul E. J. Cell Biol. 1998; 143: 319-331Crossref PubMed Scopus (119) Google Scholar, 24Linstedt A.D. Jesch S.A. Mehta A. Lee T.H. Garcia-Mata R. Nelson D.S. Sztul E. J. Biol. Chem. 2000; 275: 10196-10201Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and that p115 is required for ER to Golgi traffic of VSV-G protein (16Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (103) Google Scholar). Therefore, the anti-GM130 and anti-giantin antibodies could inhibit VSV-G protein transport by preventing p115 membrane binding. To address this issue, transport mixture (without cytosol) was supplemented with increasing amounts of anti-GM130 or anti-giantin antibodies, then added to perforated NRK cells that have been washed with 0.5 m KCl to remove membrane-associated p115. The cells were preincubated on ice for 30 min to allow the antibodies to bind cellular GM130 or giantin. Cytosol was added as the source of p115, and the cells were incubated at 32 °C for 60 min to allow p115 binding. The amount of p115 recovered with the membranes was determined by Western blotting and was quantitated relative to the amount of calnexin (used to standardize the amount of cells) present in each sample. As shown in Fig. 3 A,lanes 3–6, increasing amounts of affinity-purified anti-GM130 antibodies did not influence the binding of p115 to membranes. Equivalent amounts of bound p115 were recovered in those samples as in control samples (lane 1) or containing membranes treated with an irrelevant antibody against mannosidase II (lane 7). Cells that did not receive cytosol showed no detectable p115 (lane 2), indicating that the KCl wash removed all endogenous p115 and that cytosol was the only source of p115 in the assay. Similarly, p115 binding was observed when increasing concentrations of anti-giantin antibodies were added to the assay (Fig.3 B). No signal was observed when cells were omitted from the assay (lane 7). The effect of
Referência(s)