Cargo Can Modulate COPII Vesicle Formation from the Endoplasmic Reticulum
1999; Elsevier BV; Volume: 274; Issue: 7 Linguagem: Inglês
10.1074/jbc.274.7.4389
ISSN1083-351X
AutoresMeir Aridor, Sergei I. Bannykh, Tony Rowe, William E. Balch,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe COPII coat complex found on endoplasmic reticulum (ER)-derived vesicles plays a critical role in cargo selection. We now address the potential role of biosynthetic cargo in modulating COPII coat assembly and vesicle budding. The ER accumulation of vesicular stomatitis glycoprotein (VSV-G), a transmembrane protein, or the soluble PiZ variant of α1-antitrypsin, reduced levels of general COPII vesicle formation in vivo. Consistent with this result, conditions that prevent the export of VSV-G from the ER led to a significant inhibition of general COPII vesicle budding from ER microsomes and the export of an endogenous recycling protein p58in vitro. In contrast, synchronized export of VSV-G stimulated COPII vesicle budding both in vivo and in vitro. Under conditions where VSV-G is retained in the ER, we find that it can to be recovered in pre-budding complexes containing COPII components. These results suggest that the export of biosynthetic cargo is integrated with ER functions involved in protein folding and oligomerization. The ability of biosynthetic cargo to prevent or enhance ER export suggests that interactions of cargo with the COPII machinery contribute to the formation of vesicles budding from the ER. The COPII coat complex found on endoplasmic reticulum (ER)-derived vesicles plays a critical role in cargo selection. We now address the potential role of biosynthetic cargo in modulating COPII coat assembly and vesicle budding. The ER accumulation of vesicular stomatitis glycoprotein (VSV-G), a transmembrane protein, or the soluble PiZ variant of α1-antitrypsin, reduced levels of general COPII vesicle formation in vivo. Consistent with this result, conditions that prevent the export of VSV-G from the ER led to a significant inhibition of general COPII vesicle budding from ER microsomes and the export of an endogenous recycling protein p58in vitro. In contrast, synchronized export of VSV-G stimulated COPII vesicle budding both in vivo and in vitro. Under conditions where VSV-G is retained in the ER, we find that it can to be recovered in pre-budding complexes containing COPII components. These results suggest that the export of biosynthetic cargo is integrated with ER functions involved in protein folding and oligomerization. The ability of biosynthetic cargo to prevent or enhance ER export suggests that interactions of cargo with the COPII machinery contribute to the formation of vesicles budding from the ER. Newly synthesized (biosynthetic) cargo translocated into the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; VSV-G, vesicular stomatitis glycoprotein; TGN, trans-Golgi network; VSV, vesicular stomatitis virus; GST, glutathioneS-transferase; NRK, normal rat kidney; OKA, okadaic acid; CHX, cycloheximide; MSP, medium speed pellet; MSS, medium speed supernatant; GTPγS, guanosine 5′-3-O-(thio)triphosphate.1The abbreviations used are: ER, endoplasmic reticulum; VSV-G, vesicular stomatitis glycoprotein; TGN, trans-Golgi network; VSV, vesicular stomatitis virus; GST, glutathioneS-transferase; NRK, normal rat kidney; OKA, okadaic acid; CHX, cycloheximide; MSP, medium speed pellet; MSS, medium speed supernatant; GTPγS, guanosine 5′-3-O-(thio)triphosphate. is exported to downstream compartments by COPII vesicle carriers (reviewed in Refs. 1Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar and 2Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Incorporation into vesicles is dependent on protein folding, a process that is stringently monitored by the ER (3Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (773) Google Scholar). During export, biosynthetic cargo is sorted from resident ER proteins and selected for incorporation into vesicles by interacting with the Sar1 and Sec23/24 components of the COPII coat prior to vesicle formation (4Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar, 5Kuehn M.J. Herrmann M. Schekman R. Nature. 1998; 391: 187-190Crossref PubMed Scopus (321) Google Scholar). Subsequently, the selected cargo incorporated into these pre-budding complexes is packaged into vesicles upon addition of the Sec13/31 complex. Because both membrane-associated proteins and soluble cargo proteins become physically associated with a pre-budding complex prior to export, it becomes important to determine whether biosynthetic cargo or cargo receptors can regulate vesicle coat assembly (4Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar, 5Kuehn M.J. Herrmann M. Schekman R. Nature. 1998; 391: 187-190Crossref PubMed Scopus (321) Google Scholar), thereby linking cargo sorting to vesicle formation from the ER. A role for cargo in directing vesicle formation was previously observed in both the late secretory pathway and the endocytic pathway (reviewed in Ref. 6Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (669) Google Scholar). The transferrin receptor contains sorting determinants that are capable of interacting with the AP-2 complex that mediates clathrin-dependent endocytosis from the plasma membrane (reviewed in Ref. 7Bonifacino J.S. Marks M.S. Ohno H. Kirchhausen T. Proc. Assoc. Am. Physicians. 1996; 4: 285-295Google Scholar). Overexpression of transferrin leads to increased AP-2/clathrin coat recruitment to the plasma membrane and clathrin-coated endocytic vesicle formation (8Iacopetta B.J. Rothenberger S. Kühn L.C. Cell. 1988; 54: 485-489Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 9Millar K. Shipman M. Trowbridge I.S. Hopkins C.R. Cell. 1991; 65: 621-632Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Cargo proteins and cargo receptors in the trans-Golgi network (TGN), including mannose 6-phosphate receptors and the major histocompatibility class II protein complex, contain sorting determinants that are capable of binding to the AP-1 adaptor complex (7Bonifacino J.S. Marks M.S. Ohno H. Kirchhausen T. Proc. Assoc. Am. Physicians. 1996; 4: 285-295Google Scholar). The AP-1 complex mediates clathrin-dependent export of these proteins from the TGN (10Le Borgne R. Griffiths G. Hoflack B. J. Biol. Chem. 1996; 271: 2162-2170Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Le Borgne R. Hoflack B. J. Cell Biol. 1997; 137: 335-345Crossref PubMed Scopus (88) Google Scholar, 12Salamero J. Le Borgne R. Saudrais C. Goud B. Hoflack B. J. Biol. Chem. 1996; 271: 30318-30321Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Overexpression or elimination of these cargo proteins affects both AP-1 coat recruitment and clathrin-coated vesicle formation. To determine whether cargo exported from the ER is capable of modulating COPII vesicle formation, we have taken advantage of vesicular stomatitis virus (VSV)-infected cells to control biosynthetic cargo availability. VSV-infected cells express a single surface glycoprotein, VSV-G. The transport of both the wild-type and a mutant form of VSV-G, strain tsO45, has been extensively utilized to define the basic biochemical components and principles of operation of the secretory pathway (reviewed in Refs. 2Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 13Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1993) Google Scholar, and 14Bannykh S. Nishimura N. Balch W.E. Trends Cell Biol. 1998; 8: 21-25Abstract Full Text PDF PubMed Scopus (122) Google Scholar). In particular, tsO45 VSV-G (VSV-Gts) accumulates in the ER due to a temperature-sensitive folding defect at the restrictive temperature of 39.5 °C. Upon shift to the permissive temperature (32 °C) folding resumes, allowing us to synchronize exit from the ER. The ability to synchronize movement of VSV-Gts has played an important role in defining the normal folding intermediates associated with the molecular chaperones calnexin and BiP preceding ER export (15Hammond C. Helenius A. Science. 1994; 266: 456-458Crossref PubMed Scopus (273) Google Scholar, 16De Silva A.M. Balch W.E. Helenius A. J. Cell Biol. 1990; 111: 857-866Crossref PubMed Scopus (122) Google Scholar, 17Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (329) Google Scholar, 18Hammond C. Helenius A. Curr. Biol. 1995; 7: 523-529Crossref Scopus (586) Google Scholar). In addition, because host protein synthesis is blocked following VSV infection of cells, VSV-Gts is the major glycoprotein transiting the secretory pathway. Therefore, VSV-Gts allows us to follow the effects of a single well defined biosynthetic cargo molecule on COPII recruitment and vesicle budding when it has accumulated in the ER as a folding intermediate or when it is subsequently matured to the folded state for transport (4Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar,19Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (338) Google Scholar, 20Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 21Beckers C.J.M. Keller D.S. Balch W.E. Cell. 1987; 50: 523-534Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 22Plutner H. Davidson H.W. Saraste J. Balch W.E. J. Cell Biol. 1992; 119: 1097-1116Crossref PubMed Scopus (173) Google Scholar, 23Presley J.F. Cole N.B. Schroer T.A. Hirschberg K. Zaal K.J.M. Lippincott-Schwartz J. Nature. 1997; 389: 81-84Crossref PubMed Scopus (3) Google Scholar). We have now applied a variety of techniques to monitor the effect of biosynthetic cargo on vesicle budding from secretory compartments. These include the following: (i) quantitative stereology to follow membrane flow at the ultra-structural level in vivo (24Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (327) Google Scholar), (ii) biochemical assays to monitor ER vesicle budding in vitro (25Rowe T. Aridor M. McCaffery J.M. Plutner H. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (146) Google Scholar), (iii) an assay to monitor the interactions of cargo with COPII components in the ER prior to vesicle formation (4Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar), and (iv) an assay that allows us to follow the recruitment of COPI components to downstream pre-Golgi and Golgi compartments that may be affected by export from the ER. The effects of availability of a membrane-bound cargo molecule such as VSV-G on ER export was also compared with that of an endogenous soluble cargo molecule, the PiZ variant of α1-antitrysin (PiZ) (26Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1988; 263: 7330-7335Abstract Full Text PDF PubMed Google Scholar, 27Sifers R.N. Nat. Struct. Biol. 1995; 2: 355-357Crossref PubMed Scopus (50) Google Scholar) which accumulates in the ER under pathophysiological conditions. We now demonstrate that cargo can prevent and enhance COPII vesicle budding in vivo andin vitro. Furthermore, we show that VSV-G held in the ER interacts with the Sar1 and Sec23/24 components of the COPII machinery. We propose that the observed coupling between cargo and COPII components can modulate the formation of pre-budding intermediates that are essential for vesicle formation from the ER. Calphostin C, Roche 31-8220, staurosporine, and phorbol 12-myristate 13-acetate were obtained from Calbiochem. Calphostin C was made fresh every 2 weeks. Both reagents were added from stock solutions in dimethyl sulfoxide (Me2SO). Digitonin was obtained from Wako BioProducts (Richmond, VA). Endoglycosidase H was obtained from Boehringer Mannheim. GS beads were obtained from Amersham Pharmacia Biotech. Other antibodies used in this study were generous gifts from the following laboratories: a polyclonal antibody against Sec23p from R. Schekman, University of California, Berkeley (Berkeley, CA); a polyclonal antibody against p58 from J. Saraste, University of Bergen (Oslo, Norway); a monoclonal antibody to VSV-G from T. Kreis (28Kreis T.E. EMBO J. 1986; 5: 931-941Crossref PubMed Scopus (282) Google Scholar); a monoclonal antibody against β-COP (M3A5) from T. Kreis, University of Geneva (Geneva, Switzerland); and an anti-peptide antibody against β-COP (EAGE) from M. Farquhar (University of California, San Diego, CA). A polyclonal antibody specific for VSV-G was described previously (22Plutner H. Davidson H.W. Saraste J. Balch W.E. J. Cell Biol. 1992; 119: 1097-1116Crossref PubMed Scopus (173) Google Scholar). A polyclonal antibody to Sec23 was generated against a peptide (DTEHGGSQAR) (residues 707–716) or against recombinant GST-Sec23 as described (4Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar). The stably transfected mouse hepatoma cells (line Hep 1a) expressing recombinant PiZ variant (line H1A/RSVATZ-8) (29Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar) or normal PiM1 human AAT (line H1A/M-15) (26Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1988; 263: 7330-7335Abstract Full Text PDF PubMed Google Scholar) were generously provided by R. Sifers, Baylor College of Medicine (Houston). Measurement of the basic cell morphometric parameters, the number of ER and Golgi-derived budding profiles, and the number and size of pre-Golgi intermediates were determined as described (24Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (327) Google Scholar) Recombinant Sar1 and GST-Sec23 proteins utilized in this study were prepared as described (4Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar). Normal rat kidney (NRK) cells were infected with VSV as described (22Plutner H. Davidson H.W. Saraste J. Balch W.E. J. Cell Biol. 1992; 119: 1097-1116Crossref PubMed Scopus (173) Google Scholar, 30Davidson H.W. Balch W.E. J. Biol. Chem. 1993; 268: 4216-4226Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were infected at a multiplicity of 10–20 plaque-forming units with wild-type or the tsO45 strain of vesicular stomatitis virus (21Beckers C.J.M. Keller D.S. Balch W.E. Cell. 1987; 50: 523-534Abstract Full Text PDF PubMed Scopus (195) Google Scholar) for 4 h at the restrictive temperature. For measurement of transport in vivo, protocols were as described under "Results" and in the figure legends. For measurement of transport in vitro, preparation of cell homogenates was as described (25Rowe T. Aridor M. McCaffery J.M. Plutner H. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (146) Google Scholar). COPII vesicle formation reactions using NRK microsomes were performed and budding quantitated using antibodies specific for Sec23, p58, and VSV-G using Western blotting (25Rowe T. Aridor M. McCaffery J.M. Plutner H. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (146) Google Scholar). To enhance preservation of the temperature-sensitive phenotype observedin vivo, in some experiments (see "Results"), 1 mm dithiothreitol was added to cells during the final 15 min of incubation at 39.5 °C. Cells were harvested, washed, and homogenized in the presence of 1 mm dithiothreitol. Subsequently, membranes were washed twice in transport buffer lacking dithiothreitol prior to freezing as described(25). 150-mm dishes of NRK cells were washed three times with ice-cold phosphate-buffered saline and scraped with rubber policeman in a buffer containing 10 mm Hepes (pH 7.2) and 250 mm mannitol (buffer A). The cells were pelleted, resuspended in buffer A, and homogenized by passing the cell suspension 6 times through a ball bearing homogenizer (31Balch W.E. Rothman J.E. Arch. Biochem. Biophys. 1985; 240: 413-425Crossref PubMed Scopus (209) Google Scholar). A post-nuclear supernatant was prepared by centrifuging the homogenate at 1000 ×g for 10 min at 4 °C. 15 μl (20–40 μg protein) of post-nuclear supernatant was added to a transport reaction mixture containing 27.5 mm Hepes-KOH (pH 7.2), 2.5 mmMgOAc, 65 mm KOAc, 5 mm EGTA, 1.8 mm CaCl2, 1 mm ATP, 5 mm creatine phosphate, 0.2 units of rabbit muscle creatine kinase (final concentrations), and 1.5–2 mg/ml rat liver cytosol in a final volume of 200 μl on ice. The tubes were then transferred to 32 °C for 5–20 min as indicated under "Results." The reaction was terminated by transfer to ice. 1 ml of a buffer containing 25 mm Hepes (pH 7.2), 2.5 mm MgOAc, and KOAc to give the final salt concentration as indicated under "Results" was added, and the tubes were vortexed and centrifuged at 16,000 ×g for 10 min at 4 °C. The supernatant was aspirated, and the tubes were centrifuged for an additional 3 min at 16,000 ×g at 4 °C. The residual supernatant was removed and 25 μl of a gel sample buffer (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205498) Google Scholar) added. Samples were heated for 5 min at 95 °C and resolved using SDS-polyacrylamide gel electrophoresis (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205498) Google Scholar) on 7.5 or 10% polyacrylamide gels. β-COP was detected by immunoblotting using the M3A5 monoclonal antibody specific for β-COP and developed with alkaline phosphatase (33Duden R. Griffiths G. Frank R. Argos P. Kreis T.E. Cell. 1991; 64: 649-665Abstract Full Text PDF PubMed Scopus (362) Google Scholar, 34Pepperkok R. Scheel J. Horstmann H. Hauri H.P. Griffiths G. Kreis T.E. Cell. 1993; 74: 71-82Abstract Full Text PDF PubMed Scopus (276) Google Scholar). β-COP was quantitated by densitometry using a Molecular Dynamics Densitometer (Sunnyvale, CA). Isolation of the pre-budding complex with GST-Sec23 was performed as described (4Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar). An ER-derived bud is defined as an elevation on the surface of the ER with a width of 60–80 nm that is extruded by 50% of its diameter and covered with a characteristic coat on the external leaflet of the membrane (24Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (327) Google Scholar) (Fig.1 A, asterisks) (see "Experimental Procedures"). Such buds are found in specialized sites of the ER referred to as "transitional regions" near the Golgi (35Palade G.E. Science. 1975; 189: 347-354Crossref PubMed Scopus (2313) Google Scholar) or "export complexes" found in the peripheral cytoplasm (24Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (327) Google Scholar). To assess the effect of cargo on ER export in vivo, we examined whether the number of buds found on the ER surface can be modulated by biosynthetic cargo using electron microscopy and quantitative stereology. To validate this approach, we first determined the effect of reagents that block general protein export from the ER. We have previously established using biochemical approaches that both calphostin C and okadaic acid (OKA) are potent inhibitors of the export of the type 1 transmembrane protein vesicular stomatitis virus glycoprotein (VSV-G) from the ER, whereas phorbol 12-myristate 13-acetate is stimulatory (36Davidson H.W. McGowan C.H. Balch W.E. J. Cell Biol. 1992; 116: 1343-1355Crossref PubMed Scopus (102) Google Scholar, 37Fabbri M. Bannykh S. Balch W.E. J. Biol. Chem. 1994; 269: 26848-26857Abstract Full Text PDF PubMed Google Scholar). The effects of these reagents on the formation of ER buds as assessed by quantitative morphometry paralleled those seen in previous biochemical assays. In control cells, we detected on average 248 ± 25 buds per cell at steady state. Treatment of cells with OKA reduced by >95% the number of budding structures on the ER (12 ± 5 buds per cells). This result is similar to the recently reported morphological effects of OKA on export of the pre-Golgi intermediate marker protein p53/58 from the ER (38Pryde J.G. Farmaki T. Lucocq J.M. Mol. Cell. Biol. 1998; 18: 1125-1135Crossref PubMed Scopus (16) Google Scholar). Calphostin C led to an 85% reduction (40 ± 10 buds per cell, respectively), whereas phorbol 12-myristate 13-acetate treatment resulted in a 2-fold increase in their number (480 ± 11). Changes in ER export also led to corresponding alterations in the abundance and size of downstream pre-Golgi intermediates. These structures are composed morphologically of clusters of vesicles and tubules (Fig.1 A, arrowheads) (20Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 24Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (327) Google Scholar, 39Saraste J. Kuismanen E. Cell. 1984; 38: 535-549Abstract Full Text PDF PubMed Scopus (347) Google Scholar, 40Saraste J. Svensson K. J. Cell Sci. 1991; 100: 415-430Crossref PubMed Google Scholar) (see "Experimental Procedures"). In the absence of inhibitors, cells contained on average 67 ± 7 pre-Golgi intermediates at steady state. This value was reduced to 4 ± 1 and 14 ± 4 in the presence of OKA and calphostin C, respectively. By having established that the number of ER buds scored by quantitative morphometry serves as a reliable measure of protein and membrane flow from the ER through the secretory pathway, we next followed the effect of a single biosynthetic cargo species on ER export. For this purpose, we infected cells with the tsO45 strain of vesicular stomatitis virus (VSVts) which encodes a temperature-sensitive mutant of the type 1 transmembrane surface glycoprotein VSV-G (VSV-Gts). When expressed at the restrictive temperature (39.5 °C), VSV-Gts accumulates in the ER due to a folding defect (41Lafay F. J. Virol. 1974; 14: 1220-1228Crossref PubMed Google Scholar). Accumulation in the ER at 39.5 °C is not a consequence of aggregation as VSV-Gts is freely diffusible (42Storrie B. Pepperkok R. Stelzer E.H. Kreis T.E. J. Cell Sci. 1994; 107: 1309-1319PubMed Google Scholar). Because virus infection inhibits host protein synthesis (43Dunigan D.D. Lucas-Lenard J.M. J. Virol. 1983; 45: 618-626Crossref PubMed Google Scholar), this approach allows us to simultaneously reduce endogenous biosynthetic cargo and introduce a single major cargo species into the ER. Upon shift to the permissive temperature (32 °C), VSV-Gts exits the ER via COPII vesicular carriers (19Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (338) Google Scholar, 21Beckers C.J.M. Keller D.S. Balch W.E. Cell. 1987; 50: 523-534Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 22Plutner H. Davidson H.W. Saraste J. Balch W.E. J. Cell Biol. 1992; 119: 1097-1116Crossref PubMed Scopus (173) Google Scholar, 25Rowe T. Aridor M. McCaffery J.M. Plutner H. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (146) Google Scholar). If the availability of cargo for export can influence the formation of COPII-derived vesicular carriers in vivo, then we should detect differences between the number of ER buds and downstream intermediates found at the restrictive and permissive temperatures. We first examined the export of wild-type VSV-G to rule out the trivial concern that viral pathogenesis may interfere in some unexpected way with the appearance of budding structures. Budding activity (as defined by the number of budding profiles per μm2 ER membrane surface) in mock-infected or wild-type-infected cells at 39.5 °C was generally 2–2.5-fold greater than that observed at 32 °C (Fig.1 B). The abundance of pre-Golgi intermediates in mock-infected NRK cells was also greater (∼1.8-fold) at 39.5 °C than at 32 °C (Fig. 1 D, compare lane a tolane c), corresponding to the enhanced level of ER export. Consistent with this result, the rate of processing of wild-type VSV-G oligosaccharides by Golgi-associated enzymes was ∼2.5-fold faster at 39.5 °C than at 32 °C (data not shown). Thus, the transport activities observed in control and wild-type virus-infected cells were comparable, and in both cases, budding activity was temperature-dependent. In contrast to the lack of effects of wild-type VSV-G on budding when compared with mock-infected cells, the number of ER buds in tsO45-infected cells retained at 39.5 °C was nearly 3–4-fold lower (a 65–75% decrease) (Fig. 1 C, lane b) than that observed in mock-infected (Fig. 1 C, lane a) or wild-type VSV-infected cells (Fig. 1 B, lane b). No changes in any of the basic cell morphometric parameters (volume and surface area) were detected under these conditions (not shown). Although the surface area and volume of the ER in tsO45-infected cells held at 39.5 °C increased only slightly (∼5%), we observed an ∼50% reduction in the number of pre-Golgi intermediates (Fig. 1 D, lane b), a 40% (± 5%) decrease in the size of the Golgi stack, and a 60% (± 10%) reduction in the number of Golgi buds relative to mock-infected cells. These results suggest that the reduction in ER budding activity observed in tsO45-infected cells held at 39.5 °C (Fig. 1 C, lane b) results in a corresponding reduction in size and budding activity of downstream organelles. Because export from the ER appears to be sensitive to cargo, we would predict that transfer of tsO45-infected cells to 32 °C should restore budding activity and membrane flow. Indeed, a 5-min shift to 32 °C led to a wave of export activity, with a 1.5-fold increase (Fig. 1 C, lane d) in vesicle budding over that observed in mock-infected cells held at 32 °C (Fig. 1 B, lane c). This wave of export was reflected in a corresponding ∼1.4-fold increase in pre-Golgi intermediates (Fig. 1 D, lane d) and an ∼1.6-fold increase in Golgi size and the number of Golgi-associated buds (not shown). This value returned to the steady-state level seen in control cells within 30 min (Fig. 1 C, lane e). We conclude that a major biosynthetic cargo molecule such as VSV-Gts can affect general membrane flow from the ER by preventing or enhancing ER budding. The effects of VSV-Gts, a transmembrane protein, on export led us to also characterize the potential role of accumulation of an endogenous soluble protein, in this case the human PiZ variant of α1-antitrysin (AATPiZ) (27Sifers R.N. Nat. Struct. Biol. 1995; 2: 355-357Crossref PubMed Scopus (50) Google Scholar). Unlike wild-type monomeric α1-antitrypsin (AATwt), which is secreted rapidly from the ER, greater than 85% of the transport-impaired PiZ variant is retained within the ER and is subjected to rapid intracellular degradation (29Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar). In a subset of PiZ variant carrier patients, the degradation of the protein is markedly reduced leading to its accumulation in the ER (27Sifers R.N. Nat. Struct. Biol. 1995; 2: 355-357Crossref PubMed Scopus (50) Google Scholar). These patients develop severe liver injury, a problem that may potentially reflect a more global deficiency in ER and/or cell function. Interestingly, when we examined isogenic cell lines expressing either the wild-type α1-antitrypsin (ATTwt) or those accumulating the PiZ variant (ATTPiZ), we found an ∼30% decrease in the number of ER buds in the PiZ variant (Fig.2 A, compare lane ato lane e). Thus, steady-state accumulation of ATTPiZ in the ER led to reduced levels of budding profiles. To mimic the more exaggerated long term accumulation of α1-antitrypsin found in PiZ variant patients, we took advantage of previous studies which have demonstrated that ER degradation of the PiZ variant requires protein synthesis (44Le A. Ferrell G.A. Dishon D.S. Le Q.-Q.A. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar). Addition of CHX not only prevents degradation of the PiZ variant but also reduces the content of other cargo proteins in the ER. As such, this condition leads to a temporary relative increase in the abundance of polymerized AATPiZ variant in the ER (44Le A. Ferrell G.A. Dishon D.S. Le Q.-Q.A. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar, 45Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 267: 1072-1080Google Scholar). In addition, incubation of cells in the presence of CHX allows us to examine independently the role of general cargo reduction on ER-budding structures. This is an important control in the case of VSV-infected cells where the virus inhibits host protein synthesis. Previous studies in yeast have shown that CHX treatment does lead to a partial reduction of ER-derived vesicles (46Kaiser C.A. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (536) Google Scholar). To test first for any potential nonspecific effects of CHX on transport, we examined whether exposure of tsO45 virus-infected cells to CHX for up to 4 h at the restrictive temperature alters the subsequent kinetics of ER to Golgi transport of VSV-Gts at the permissive temperature in the presence of the drug. As shown in Fig. 2 B, VSV-G accumulated at the restrictive temperature for 4.5 h was radiolabeled at 39.5 °C and incubated in the presence of CHX for an additional 4 h prior to transfer to 32 °C. Strikingly, no inhibitory effect was observed compared with the transport kinetics of the control that was not incubated for an additional 4 h in the presence of CHX. A similar lack of effe
Referência(s)