Membrane Recruitment of Coatomer and Binding to Dilysine Signals Are Separate Events
2000; Elsevier BV; Volume: 275; Issue: 37 Linguagem: Inglês
10.1074/jbc.m003630200
ISSN1083-351X
AutoresMarie Gomez, Suzie J. Scales, Thomas E. Kreis, Franck Perez,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoIt has previously been shown that transport of newly synthesized proteins and the structure of the Golgi complex are affected in the Chinese hamster ovary cell line ldlF, which bears a temperature-sensitive mutation in the Coat protein I (COPI) subunit ε-COP (Guo, Q., Vasile, E., and Krieger, M. (1994)J. Cell Biol. 125, 1213–1224; Hobbie, L., Fisher, A. S., Lee, S., Flint, A., and Krieger, M. (1994) J. Biol. Chem. 269, 20958–20970). Here, we pinpoint the site of the secretory block to an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi complex and show that the distributions of ER-Golgi recycling proteins, such as KDEL receptor and p23, as well as resident Golgi proteins, such as mannosidase II, are accordingly affected. At the nonpermissive temperature, neither the stability of the COPI complex nor its recruitment to donor Golgi membranes is affected. However, the binding of coatomer to the dilysine-based ER-retrieval motif is impaired in the absence of ε-COP, suggesting that dilysine signal binding is not the major means of COPI recruitment. Because expression of the exogenous chimera of ε-COP and green fluorescent protein in ldlF cells at nonpermissive temperature rapidly restores the wild type properties, ε-COP is likely to play an important role in the cargo selection events mediated by COPI. It has previously been shown that transport of newly synthesized proteins and the structure of the Golgi complex are affected in the Chinese hamster ovary cell line ldlF, which bears a temperature-sensitive mutation in the Coat protein I (COPI) subunit ε-COP (Guo, Q., Vasile, E., and Krieger, M. (1994)J. Cell Biol. 125, 1213–1224; Hobbie, L., Fisher, A. S., Lee, S., Flint, A., and Krieger, M. (1994) J. Biol. Chem. 269, 20958–20970). Here, we pinpoint the site of the secretory block to an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi complex and show that the distributions of ER-Golgi recycling proteins, such as KDEL receptor and p23, as well as resident Golgi proteins, such as mannosidase II, are accordingly affected. At the nonpermissive temperature, neither the stability of the COPI complex nor its recruitment to donor Golgi membranes is affected. However, the binding of coatomer to the dilysine-based ER-retrieval motif is impaired in the absence of ε-COP, suggesting that dilysine signal binding is not the major means of COPI recruitment. Because expression of the exogenous chimera of ε-COP and green fluorescent protein in ldlF cells at nonpermissive temperature rapidly restores the wild type properties, ε-COP is likely to play an important role in the cargo selection events mediated by COPI. coat protein ADP-ribosylation factor brefeldin A green fluorescent protein glutathione S-transferase transport complex temperature-sensitive mutant of the glycoprotein of vesicular stomatitis virus Chinese hamster ovary endoplasmic reticulum phosphate-buffered saline antibody polyacrylamide gel electrophoresis Coated vesicles participate in sorting and transport of newly synthesized proteins in the early secretory pathway. To date, two coat protein (COP)1 complexes, COPI and COPII, have been identified that mediate transport between the endoplasmic reticulum (ER) and the Golgi complex (1Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar, 2Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1021) Google Scholar, 3Scales S.J. Gomez M. Kreis T.E. Int. Rev. Cytol. 2000; 95: 67-144Google Scholar). There is strong evidence for COPII mediating anterograde transport from the ER to the ER-Golgi intermediate compartment, in mammals (4Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (340) Google Scholar, 5Scales S.J. Pepperkok R. Kreis T.E. Cell. 1997; 90: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), or to the Golgi, in yeast (6Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 7Bednarek S.Y. Ravazzola M. Hosobuchi M. Amherdt M. Perrelet A. Schekman R. Orci L. Cell. 1995; 83: 1183-1196Abstract Full Text PDF PubMed Scopus (230) Google Scholar). However, the involvement of COPI in anterograde and/or retrograde transport both between the ER and the Golgi complex and within the Golgi complex is highly debated, and experiments supporting all of these functions have been reported (reviewed in Refs. 3Scales S.J. Gomez M. Kreis T.E. Int. Rev. Cytol. 2000; 95: 67-144Google Scholar and 8Cosson P. Letourneur F. Curr. Opin. Cell Biol. 1997; 9: 484-487Crossref PubMed Scopus (119) Google Scholar).It has been proposed that the COPI coat mediates sorting and incorporation of cargo molecules into nascent vesicles through interactions with sorting determinants in the cytoplasmic tails of cargo proteins (2Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1021) Google Scholar). Several sorting signals have been identified in the early biosynthetic pathway between the Golgi complex and the ER, including the KKXX and KXKXX, or dilysine, retrieval motifs for integral membrane proteins and KDEL for soluble lumenal proteins of the ER (reviewed in Ref. 9Teasdale R.D. Jackson M.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 27-54Crossref PubMed Scopus (442) Google Scholar). A direct interaction has been demonstrated for KKXX-containing proteins with COPI in vitro (10Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (480) Google Scholar), and this interaction may be physiological because certain COPI mutants in yeast are defective in retrieval of KKXX- and KDEL-bearing proteins from the Golgi (11Letourneur F. Gaynor E.C. Hennecke S. Demolliere C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 12Lewis M.J. Pelham H.R. Cell. 1996; 85: 205-215Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Putative anterograde signals include the diphenylalanine (FF) motif (13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar) and the DXE motif (14Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (394) Google Scholar), although the latter appears to mediate concentration rather than sorting per se(15Nishimura N. Bannykh S. Slabough S. Matteson J. Altschuler Y. Hahn K. Balch W.E. J. Biol. Chem. 1999; 274: 15937-15946Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The p24 family of integral membrane proteins are the most abundant of the dilysine- and diphenylalanine-containing proteins of the early secretory pathway (13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar, 16Rojo M. Pepperkok R. Emery G. Kellner R. Stang E. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1119-1135Crossref PubMed Scopus (122) Google Scholar, 17Dominguez M. Dejgaard K. Füllekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J.M. Nillso T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (289) Google Scholar), but it is still a debate whether the cytoplasmic tail of p24-related proteins represent the major binding site for COPI complex on donor membranes (13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar,18Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (180) Google Scholar, 19Nickel W. Sohn K. Bunning C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11393-11398Crossref PubMed Scopus (65) Google Scholar, 20Pavel J. Harter C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2140-2145Crossref PubMed Scopus (69) Google Scholar, 21Bremser M. Nickel W. Schweikert M. Ravazzola M. Amherdt M. Hughes C.A. Söllner T.H. Rothman J.E. Wieland F.T. Cell. 1999; 96: 495-506Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 22Rojo M. Emery G. Marjomaki V. McDowall A.W. Parton R.G. Gruenberg J. J. Cell Sci. 2000; 113: 1043-1057Crossref PubMed Google Scholar).Although the general mechanism of COPI-coated vesicle formation is quite well described (1Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar, 2Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1021) Google Scholar), the specific roles of the individual COPI subunits in this process are not yet clear. COPI consists of coatomer, a complex composed of seven subunits (α-, β-, β′-, γ-, δ-, ε-, and ζ-COP (23Waters M.G. Serafini T. Rothman J.E. Nature. 1991; 349: 248-251Crossref PubMed Scopus (375) Google Scholar, 24Stenbeck G. Schreiner R. Herrmann D. Auerbach S. Lottspeich F. Rothman J.E. Wieland F.T. FEBS Lett. 1992; 314: 195-198Crossref PubMed Scopus (42) Google Scholar)) and the small GTP-binding protein ARF, which may also be considered as a component of the coat (25Serafini T. Orci L. Amherdt M. Brunner M. Kahn R.A. Rothman J.E. Cell. 1991; 67: 239-253Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 26Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (328) Google Scholar, 27Stamnes M.A. Craighead M.W. Hoe M.H. Lampen N. Geromanos S. Tempst P. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8011-8015Crossref PubMed Scopus (195) Google Scholar).In vivo, the assembly of coatomer takes about 1–2 h, with ε-COP being the last subunit to be incorporated. Once formed, coatomer is very stable, with a half-life of ∼28 h and no apparent subunit exchange with newly synthesized COPs (28Lowe M. Kreis T.E. J. Biol. Chem. 1996; 271: 30725-30730Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In vitro, coatomer can be reversibly disassembled into subcomplexes of α/ε/β′, β/δ, and γ/ζ (29Lowe M. Kreis T.E. J. Biol. Chem. 1995; 270: 31364-31371Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and these interactions have been confirmed by two-hybrid analysis (30Faulstich D. Auerbach S. Orci L. Ravazzola M. Wegchingel S. Lottspeich F. Stenbeck G. Harter C. Wieland F.T. Tschochner H. J. Cell Biol. 1996; 135: 53-61Crossref PubMed Scopus (76) Google Scholar). It has been proposed that different parts of COPI bind to distinct cytosolic signals to mediate sorting in different directions, such as α/ε/β′ binding to dilysine motifs for retrograde transport (10Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (480) Google Scholar, 13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar, 29Lowe M. Kreis T.E. J. Biol. Chem. 1995; 270: 31364-31371Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and β/γ/ζ binding to FF motifs for anterograde transport in the secretory pathway (13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar). However, other studies have shown that γ-COP can be cross-linked to KKXX motifs (31Harter C. Pavel J. Coccia F. Draken E. Wegehingel S. Tschochner H. Wieland F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1902-1906Crossref PubMed Scopus (79) Google Scholar), and indirect evidence suggests that coatomer has at least two dilysine binding sites (32Hudson R.T. Draper R.K. Mol. Biol. Cell. 1997; 8: 1901-1910Crossref PubMed Scopus (29) Google Scholar).A CHO cell line (ldlF) with a temperature-sensitive mutation in ε-COP (33Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar) has been shown to exhibit defects in the transport of newly synthesized proteins and in the maintenance of Golgi structure at the nonpermissive temperature (33Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar, 34Hobbie L. Fisher A.S. Lee S. Flint A. Krieger M. J. Biol. Chem. 1994; 269: 20958-20970Abstract Full Text PDF PubMed Google Scholar). At nonpermissive temperature, ε-COP is rapidly degraded without affecting the levels of the other subunits, except for an ∼2-fold reduction in α-COP (35Guo Q. Penman M. Trigatti B.L. Krieger M. J. Biol. Chem. 1996; 271: 11191-11196Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Gu F. Aniento F. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (138) Google Scholar). These phenotypes are corrected by stably transfecting wild type ε-COP (33Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar,34Hobbie L. Fisher A.S. Lee S. Flint A. Krieger M. J. Biol. Chem. 1994; 269: 20958-20970Abstract Full Text PDF PubMed Google Scholar, 37Shima D.T. Scales S.J. Kreis T.E. Pepperkok R. Curr. Biol. 1999; 9: 821-824Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). We have used this ldlF cell line as a tool to examine the specific role of ε-COP in the COPI complex in the biosynthetic pathway. We find that at nonpermissive temperature, transport to the plasma membrane of the temperature-sensitive mutant of the glycoprotein of vesicular stomatitis virus (ts-045-G) is blocked in an ER/Golgi intermediate compartment. In addition, we show that the distributions of recycling markers, such as KDEL receptor and p23, or resident Golgi proteins, such as mannosidase II, are also affected. At nonpermissive temperature, neither the stability of the COPI complex nor its recruitment to donor membranes is altered. However, the binding of coatomer to the dilysine ER-retrieval motif is impaired, indicating that coatomer recruitment to donor membranes and cargo selection are not identical but separate events. Because microinjection and expression of ε-COP-GFP cDNA in ldlF cells at nonpermissive temperature rapidly restores secretory function and Golgi structure, ε-COP is most likely involved in sorting/retrieval events in the early secretory pathway between the ER and Golgi and in the maintenance of Golgi structure.DISCUSSIONWe have taken advantage of the existence of a temperature-sensitive mammalian cell line (ldlF CHO cells) in which one of the coatomer subunits, ε-COP, is degraded at nonpermissive temperature to study the possible role played by this subunit in coatomer function. Our results suggest that ε-COP is not directly involved in the stability of the coatomer complex or in its recruitment to donor membranes. However, although coatomer still exists in the cells as a membrane-bound complex in the absence of ε-COP, transport from the ER to the plasma membrane of ts-045-G protein was blocked in an ER/Golgi intermediate compartment or in transport complexes. These results show that coatomer stability and recruitment to donor membranes are not sufficient for its proper function. In addition, we could show that, in the absence of ε-COP, binding of coatomer to the dilysine ER-retrieval motif was inhibited in vitro and that the distributions of the KDEL receptor (known to be involved in the retrieval of ER-resident proteins from the intermediate compartment and the Golgi complex) and the dilysine-containing protein p23 were alteredin vivo. At nonpermissive temperature, these proteins were redistributed to membranous structures to which the other non-ε-COP subunits of coatomer were bound. Because exogenously expressed ε-COP-GFP cDNA in ldlF cells at nonpermissive temperature rapidly colocalized with endogenous coatomer and restored all of the wild type properties, we conclude that ε-COP plays an important role in coatomer function, probably directly participating in sorting/retrieval events.Exactly how coat proteins are recruited to specific sites on the donor membrane to initiate budding is still unclear. Several lines of evidence suggest that cargo itself could stimulate coated vesicle formation. For example, the knockout in mice of the two mannose-6-phosphate receptors, the major cargoes of trans-Golgi network-derived clathrin-coated vesicles, results in decreased recruitment of AP-1 adaptors and clathrin to trans-Golgi network membranes (48Le Borgne R. Griffiths G. Hoflack B. J. Biol. Chem. 1996; 271: 2162-2170Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Furthermore, the binding of the plasma membrane AP-2 adaptors to tyrosine-based motifs was shown to be strengthened upon clathrin coat formation, suggesting that cargo recognition and coat assembly are indeed coupled (49Rapoport I. Miyazaki M. Boll W. Duckworth B. Cantley L.C. Shoelson S. Kirchhausen T. EMBO J. 1997; 16: 2240-2250Crossref PubMed Scopus (182) Google Scholar). By analogy, it has been proposed that some members of the p24 family, which can bind via their dilysine motifs to coatomer in vitro and are found in COPI-coated vesicles (13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar, 18Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (180) Google Scholar, 27Stamnes M.A. Craighead M.W. Hoe M.H. Lampen N. Geromanos S. Tempst P. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8011-8015Crossref PubMed Scopus (195) Google Scholar), may act in the donor membrane as cargo receptors and coatomer and/or ARF receptors. In fact, p23 and p24 cytoplasmic tails attached to artificial liposomes of physiological lipid composition appear sufficient to recruit coatomer in the presence of ARF-GTP to these membranes (21Bremser M. Nickel W. Schweikert M. Ravazzola M. Amherdt M. Hughes C.A. Söllner T.H. Rothman J.E. Wieland F.T. Cell. 1999; 96: 495-506Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). However, our results suggest that in ldlF cells at nonpermissive temperature, coatomer can still be recruited to membranes in vitro and in vivo,despite impaired binding to dilysine motifs, consistent with the fact that coatomer can also bind to Golgi membranes via ARF-GTP, probably through the β/δ subunits (20Pavel J. Harter C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2140-2145Crossref PubMed Scopus (69) Google Scholar). It seems unlikely, then, that the members of the p24 family act as the sole receptors for coatomer. It has actually been shown recently that p24 family proteins may be involved in protein sorting by modulating the COPI stimulation of the ARF GTPase activity (50Goldberg J. Cell. 2000; 100: 671-679Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), which is in agreement with the observed binding of ARF to coatomer in close proximity of the dilysine-binding domain (51Zhao L. Helms J.B. Brunner J. Wieland F.T. J. Biol. Chem. 1999; 274: 14198-14203Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Interestingly, Zhu et al. (52Zhu Y. Traub L.M. Kornfeld S. Mol. Biol. Cell. 1999; 10: 537-549Crossref PubMed Scopus (44) Google Scholar) recently reported that, similarly, mannose 6-phosphate receptor tails are not essential determinants in the initial steps of AP-1 binding to the trans-Golgi network but are most likely involved in sorting steps by modulating ARF GTPase activity. COPI complex may thus be recruited on Golgi membranes via ARF in its GTP bound form, and p24 family proteins may then contribute to stabilize or destabilize this primary complex due to particular sequences in their dilysine-containing (or diphenylalanine-containing) tail.Although we showed that ε-COP plays a major role in sorting, we did not demonstrate a direct interaction between ε-COP and KKXX motifs, and we thus do not exclude the possibility that in the absence of ε-COP, binding to KKXX motifs was altered through the other subunits of the α/β′/ε subcomplex that is thought to mediate binding to KKXX motifs. Indeed, yeast genetic studies have shown that coatomer from α-COP and β′-COP mutants has lost the ability to bind dilysine motifs in vitro. (11Letourneur F. Gaynor E.C. Hennecke S. Demolliere C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (663) Google Scholar). Also, the KDEL receptor, although it is a well established cargo of COPI-coated vesicles, has not been shown to interact directly with coatomer (18Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (180) Google Scholar, 53Orci L. Stamnes M. Ravazzola M. Amherdt M. Perrelet A. Sollner T.H. Rothman J.E. Cell. 1997; 90: 335-349Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). However, our observation that in the absence of ε-COP KDEL receptor redistributes into similar structures to the dilysine-containing p23 suggests that they use similar recycling mechanisms, perhaps implicating ε-COP. This is consistent with genetic experiments performed in yeast, implicating COPI in retrieval of both KKXX- and KDEL-containing proteins (11Letourneur F. Gaynor E.C. Hennecke S. Demolliere C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 12Lewis M.J. Pelham H.R. Cell. 1996; 85: 205-215Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 54Banfield D.K. Lewis M.J. Pelham H.R. Nature. 1995; 375: 806-809Crossref PubMed Scopus (129) Google Scholar).In ldlF cells at nonpermissive temperature, not only the sorting of recycling proteins but also that of secretory proteins was perturbed. Transport to the plasma membrane of newly synthesized ts-045-G protein was indeed blocked in structures that were positive for β′-COP, and most likely also for KDEL receptor and p23. Similarly, we have previously shown that microinjection of antibodies against a synthetic peptide of β-COP, EAGE, blocks transport of ts-045-G to the cell surface, at the interface of the ER and the Golgi, in tubular structures containing ERGIC-53 (55Pepperkok 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), suggesting that ts-045-G protein may be arrested in similar compartments in ldlF cells at nonpermissive temperature and in EAGE-microinjected cells. These structures might represent the TCs recently described in Vero cells (5Scales S.J. Pepperkok R. Kreis T.E. Cell. 1997; 90: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), although we cannot exclude the possibility that ts-045-G is arrested in the Golgi, which is also scattered at nonpermissive temperature in ldlF cells (33Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar). A model was proposed (5Scales S.J. Pepperkok R. Kreis T.E. Cell. 1997; 90: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 56Lowe M. Kreis T.E. Biochim. Biophys. Acta. 1998; 1404: 53-66Crossref PubMed Scopus (81) Google Scholar) in which COPII-coated vesicles form from the ER, cluster into vesicular tubular structures, as proposed by Balch and co-workers (57Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (332) Google Scholar, 58Bannykh S.I. Balch W.E. J. Cell Biol. 1997; 138: 1-4Crossref PubMed Scopus (192) Google Scholar), and fuse to form TCs. Subsequently, COPI replaces COPII, and the TCs to interact with microtubules and move toward the Golgi complex (5Scales S.J. Pepperkok R. Kreis T.E. Cell. 1997; 90: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 37Shima D.T. Scales S.J. Kreis T.E. Pepperkok R. Curr. Biol. 1999; 9: 821-824Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In this model, COPI is required to retrieve material to the ER from the TC and the Golgi complex. Our observations of the phenotypes of ldlF cells are consistent with this model: we showed that coatomer in ldlF cells at nonpermissive temperature loses the ability to bind dilysine motifsin vitro, which could explain several of the defects we observed in these cells. If the steady state localizations of KDEL receptor and p23 require proper recycling between the Golgi complex and/or between the intermediate compartment and the ER, the inhibition of coatomer binding to motifs required for sorting and retrieval of these proteins would alter their distributions, as we observed. It could also explain the arrest of ts-045-G protein in an intermediate compartment between the ER and the Golgi (perhaps the TC); if the retrieval function of COPI is inhibited in ldlF cells at nonpermissive temperature, factors required for earlier steps in the secretory pathway (e.g. v-SNARES, ERGIC-53, and COPII-associated proteins) would not be removed, and transfer of cargo to the Golgi would be impaired. One of the proposed roles for the abundant p23 protein is that it contributes to the structure of the intermediate compartment (16Rojo M. Pepperkok R. Emery G. Kellner R. Stang E. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1119-1135Crossref PubMed Scopus (122) Google Scholar, 22Rojo M. Emery G. Marjomaki V. McDowall A.W. Parton R.G. Gruenberg J. J. Cell Sci. 2000; 113: 1043-1057Crossref PubMed Google Scholar); therefore, missorting of p23 induced by degradation of ε-COP could potentially explain the structural changes observed in the membranous compartments of the early biosynthetic pathway.The reasons for the observed distribution of the Golgi enzyme mannosidase II in ldlF cells at nonpermissive temperature are less clear. To some extent, it is reminiscent of the effect observed with the drug BFA, the earliest detected effect of which is the removal of β-COP from Golgi membranes, resulting in the extension of tubules from the Golgi to the ER and the subsequent disappearance of the Golgi, with the redistribution of Golgi membrane proteins to the ER (59Lippincott-Schwartz J. Donaldson J.G. Schweizer A. Berger E.G. Hauri H.P. Yuan L.C. Klausner R.D. Cell. 1990; 60: 821-836Abstract Full Text PDF PubMed Scopus (727) Google Scholar, 60Strous G.J. Berger E.G. van Kerkhof P. Bosshart H. Berger B. Geuze H.J. J. Biol. Cell. 1991; 71: 25-31Crossref Scopus (32) Google Scholar). In ldlF cells at nonpermissive temperature, Golgi structure has been shown to be dramatically altered, dissociating into vesicles and tubules (33Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar). However, we observed that in ldlF cells at nonpermissive temperature, mannosidase II was still associated with patches positive for β′-COP that were not observed in ldlF cells at permissive or nonpermissive temperature after BFA treatment. This is consistent with the observation that dissociation of coatomer from membranes is required for BFA-induced transfer of Golgi enzymes to the endoplasmic reticulum (61Scheel J. Pepperkok R. Lowe M. Griffiths G. Kreis T.E. J. Cell Biol. 1997; 137: 319-333Crossref PubMed Scopus (72) Google Scholar). It is thus possible that the membrane-bound ε-COP-depleted coatomer, although it lost at least part of its sorting capacity, conserved its ability to prevent premature fusion of transport intermediate. An extension of the transport model mentioned above predicts not only that TCs mature as they travel toward the Golgi complex but also that they could potentially fuse with each other to form the cis-Golgi network (5Scales S.J. Pepperkok R. Kreis T.E. Cell. 1997; 90: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 62Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (330) Google Scholar). Because we have shown that TC maturation is blocked by the inactivation of ε-COP (this work and Ref. 5Scales S.J. Pepperkok R. Kreis T.E. Cell. 1997; 90: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), this could also explain the observed changes in Golgi structure. This is consistent with recent data showing COPI to be necessary for the in vitro formation of vesiculo-tubular clusters (TC precursors) from transitional ER microsomes (63Lavoie C. Paiement J. Dominguez M. Roy L. Dahan S. Gushue J.N. Bergeron J.J.M. J. Cell Biol. 1999; 146: 285-299Crossref PubMed Scopus (79) Google Scholar).In conclusion, although a coatomer complex is still able to form and be recruited to membranes in ldlF cells at nonpermissive temperature, secretory function, protein sorting, and Golgi structure are affected. Exogenously expressed ε-COP-GFP, which rapidly incorporates into a nonfunctional coatomer devoid of ε-COP, restores all of the wild type properties, suggesting that ε-COP is essential for the proper functioning of coatomer. In addition, the ability of ε-COP-deficient coatomer to bind membranes without binding to KKXX motifs suggests that coatomer recruitment occurs prior to cargo selection rather than being directly coupled to it. Coated vesicles participate in sorting and transport of newly synthesized proteins in the early secretory pathway. To date, two coat protein (COP)1 complexes, COPI and COPII, have been identified that mediate transport between the endoplasmic reticulum (ER) and the Golgi complex (1Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar, 2Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1021) Google Scholar, 3Scales S.J. Gomez M. Kreis T.E. Int. Rev. Cytol. 2000; 95: 67-144Google Scholar). There is strong evidence for COPII mediating anterograde transport from the ER to the ER-Golgi in
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