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• W1966398753 abstract "Further investigation of the targeting of the intracellular membrane lectin endoplasmic reticulum (ER)-Golgi intermediate compartment-53 (ERGIC-53) by site-directed mutagenesis revealed that its lumenal and transmembrane domains together confer ER retention. In addition we show that the cytoplasmic domain is required for exit from the ER indicating that ERGIC-53 carries an ER-exit determinant. Two phenylalanines at the C terminus are essential for ER-exit. Thus, ERGIC-53 contains determinants for ER retention as well as anterograde transport which, in conjunction with a dilysine ER retrieval signal, control the continuous recycling of ERGIC-53 in the early secretory pathway. In vitro binding studies revealed a specific phenylalanine-dependent interaction between an ERGIC-53 cytosolic tail peptide and the COPII coat component Sec23p. These results suggest that the ER-exit of ERGIC-53 is mediated by direct interaction of its cytosolic tail with the Sec23p·Sec24p complex of COPII and that protein sorting at the level of the ER occurs by a mechanism similar to receptor-mediated endocytosis or Golgi to ER retrograde transport. Further investigation of the targeting of the intracellular membrane lectin endoplasmic reticulum (ER)-Golgi intermediate compartment-53 (ERGIC-53) by site-directed mutagenesis revealed that its lumenal and transmembrane domains together confer ER retention. In addition we show that the cytoplasmic domain is required for exit from the ER indicating that ERGIC-53 carries an ER-exit determinant. Two phenylalanines at the C terminus are essential for ER-exit. Thus, ERGIC-53 contains determinants for ER retention as well as anterograde transport which, in conjunction with a dilysine ER retrieval signal, control the continuous recycling of ERGIC-53 in the early secretory pathway. In vitro binding studies revealed a specific phenylalanine-dependent interaction between an ERGIC-53 cytosolic tail peptide and the COPII coat component Sec23p. These results suggest that the ER-exit of ERGIC-53 is mediated by direct interaction of its cytosolic tail with the Sec23p·Sec24p complex of COPII and that protein sorting at the level of the ER occurs by a mechanism similar to receptor-mediated endocytosis or Golgi to ER retrograde transport. The early secretory pathway of higher eukaryotes includes the endoplasmic reticulum (ER), 1The abbreviations used are: ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; endo H, endo-β-N-acetylglucosaminidase H; mAb, monoclonal antibody.1The abbreviations used are: ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; endo H, endo-β-N-acetylglucosaminidase H; mAb, monoclonal antibody.the ER-Golgi intermediate compartment (ERGIC), and the Golgi apparatus that are connected by vesicle-mediated anterograde and retrograde protein transport pathways (1Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 2Bednarek S.Y. Orci L. Schekman R. Trends Cell Biol. 1996; 6: 468-473Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 3Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar, 4Bannykh S.I. Balch W.E. J. Cell Biol. 1997; 138: 1-4Crossref PubMed Scopus (192) Google Scholar, 5Farquhar M.G. Hauri H.-P. Berger E.G. Roth J. The Golgi Apparatus. Birkhäuser Verlag, Basel1997: 63-130Crossref Google Scholar, 6Hauri H.-P. Schweizer A. Hoffman J.F. Jamieson J.D. Handbook of Physiology: Cell Physiology. Oxford University Press for the American Society for Cell Biology, Oxford1997: 605-647Google Scholar, 7Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). Vesicle budding is driven by the recruitment of cytosolic coat proteins to the donor membrane resulting in the formation of a coated vesicle. Two different cytosolic coat complexes, COPI and COPII, are involved in the formation of at least two distinct classes of transport vesicles (3Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar). COPII mediates vesicle budding from the ER for anterograde protein transport (8Barlowe 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, 9Shaywitz D.A. Orci L. Ravazzola M. Swaroop A. Kaiser C.A. J. Cell Biol. 1995; 128: 769-777Crossref PubMed Scopus (85) Google Scholar, 10Paccaud J.-P. Reith W. Carpentier J.-L. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: 1535-1546Crossref PubMed Scopus (96) Google Scholar, 11Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (253) Google Scholar, 12Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (340) Google Scholar), whereas COPI has been implicated in many different traffic pathways including exit from the ER (13Peter F. Plutner H. Zhu H. Kreis T.E. Balch W.E. J. Cell Biol. 1993; 122: 1155-1167Crossref PubMed Scopus (102) Google Scholar, 14Bednarek 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), ERGIC to Golgi (15Pepperkok 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), through the Golgi (16Rothman J.E. Orci L. Nature. 1992; 355: 409-415Crossref PubMed Scopus (741) Google Scholar), early to late endosomes (17Whitney J.A. Gomez M. Sheff D. Kreis T.E. Mellman I. Cell. 1995; 83: 703-713Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 18Aniento F. Feng G. Parton R.G. Gruenberg J. J. Cell Biol. 1996; 133: 29-41Crossref PubMed Scopus (314) Google Scholar), and Golgi to ER retrograde transport by direct interaction with dilysine ER-retrieval signals (19Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (480) Google Scholar, 20Letourneur F. Gaynor E.C. Hennecke S. Démollière C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (664) Google Scholar, 21Cosson P. Démollière C. Hennecke S. Duden R. Letourneur F. EMBO J. 1996; 15: 1792-1798Crossref PubMed Scopus (124) Google Scholar, 22Pelham H.R.B. Cell. 1994; 79: 1125-1127Abstract Full Text PDF PubMed Scopus (106) Google Scholar, 23Gaynor E.C. Emr S.D. J. Cell Biol. 1997; 136: 789-802Crossref PubMed Scopus (164) Google Scholar, 24Rowe T. Aridor M. McCaffery J.M. Plutner H. Nuoffer C. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (147) Google Scholar).It is currently unclear which transport pathways directly depend on COPI. Recent in vitro binding studies proposed a bimodal interaction of two COPI subcomplexes with different cytosolic tail sequences of proteins of the p24 family (25Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar). The B-subcomplex (consisting of α-, β′-, and ε-COP) specifically binds to dilysine ER-retrieval signals (19Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (480) Google Scholar), whereas binding of the F-subcomplex (consisting of β-, γ-, and ζ-COP) requires the presence of a critical phenylalanine that may be part of an anterograde transport signal (25Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar). Based on these findings a conformational switch mechanism was proposed by which coatomer may differentiate between anterograde and retrograde transport.There is growing evidence that exit from the ER is selective rather than by default (26Pfeffer S.R. Rothman J.E. Annu. Rev. Biochem. 1987; 56: 829-852Crossref PubMed Scopus (619) Google Scholar) as indicated by the fact that properly folded secretory (27Mizuno M. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5732-5736Crossref PubMed Scopus (114) Google Scholar) and membrane (1Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 28Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (332) Google Scholar, 29Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (330) Google Scholar, 30Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1021) Google Scholar) proteins are concentrated at this step. In line with this notion, a di-acidic signal in the cytoplasmic tail of vesicular stomatitis virus glycoprotein and other membrane proteins was recently shown to be required for selective export from the endoplasmic reticulum (31Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (394) Google Scholar). It is envisaged now that sorting at the level of the endoplasmic reticulum is accomplished by sorting receptors that bind cargo proteins as well as cytosolic coat complexes (1Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (80) Google Scholar). A candidate sorting receptor for a subset of proteins in yeast is Emp24p, a major membrane protein of ER-derived COPII vesicles (32Schimmöller F. Singer-Krüger B. Schröder S. Krüger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (282) Google Scholar). Emp24p belongs to a growing family of related 24-kDa proteins also found in mammalian cells (33Wada I. Rindress D. Cameron P.H. Ou W.J. Doherty II, J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar, 34Stamnes 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, 35Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 36Sohn 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), but the suggestion that they can transport cargo as cargo receptors remains to be proven.The ERGIC marker protein ERGIC-53 (p58 in rat, Ref. 37Lahtinen U. Hellman U. Wernstedt C. Saraste J. Pettersson R.F. J. Biol. Chem. 1996; 271: 4031-4037Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) is another candidate cargo receptor (6Hauri H.-P. Schweizer A. Hoffman J.F. Jamieson J.D. Handbook of Physiology: Cell Physiology. Oxford University Press for the American Society for Cell Biology, Oxford1997: 605-647Google Scholar). ERGIC-53 is a type I membrane protein (38Hauri H.-P. Schweizer A. Curr. Opin. Cell Biol. 1992; 4: 600-608Crossref PubMed Scopus (171) Google Scholar), carries a dilysine ER-retrieval signal in its cytoplasmic domain (39Schindler R. Itin C. Zerial M. Lottspeich F. Hauri H.-P. Eur. J. Cell Biol. 1993; 61: 1-9PubMed Google Scholar), and constitutively recycles between ER, ERGIC, and cis-Golgi (39Schindler R. Itin C. Zerial M. Lottspeich F. Hauri H.-P. Eur. J. Cell Biol. 1993; 61: 1-9PubMed Google Scholar, 40Lippincott-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, 41Itin C. Foguet M. Kappeler F. Klumperman J. Hauri H.-P. Biochem. Soc. Trans. 1995; 23: 541-544Crossref PubMed Scopus (16) Google Scholar). The protein exhibits properties of a mannose-selective and calcium-dependent lectin due to a lectin domain on its lumenal side (42Fiedler K. Simons K. Cell. 1994; 77: 625-626Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 43Arar C. Carpentier V. Le Caer J.-P. Monsigny M. Legrand A. Roche A.-C. J. Biol. Chem. 1995; 270: 3551-3553Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 44Itin C. Roche A.-C. Monsigny M. Hauri H.-P. Mol. Biol. Cell. 1996; 7: 483-493Crossref PubMed Scopus (153) Google Scholar). It is conceivable therefore that ERGIC-53 binds newly synthesized glycoproteins in the ER and recruits them into budding vesicles. When the dilysine signal is inactivated, intracellular transport of ERGIC-53 in COS-1 cells remains slow indicating that a second determinant can also function in intracellular targeting (45Itin C. Schindler R. Hauri H.-P. J. Cell Biol. 1995; 131: 57-67Crossref PubMed Scopus (101) Google Scholar). The targeting efficiency of both signals is weakened by two C-terminal phenylalanines. It was suggested that the second targeting determinant is formed by the cytosolic sequence RSQQE adjacent to the transmembrane domain in conjunction with the lumenal domain of ERGIC-53 and that it may be responsible for the concentration of ERGIC-53 in the intermediate compartment (45Itin C. Schindler R. Hauri H.-P. J. Cell Biol. 1995; 131: 57-67Crossref PubMed Scopus (101) Google Scholar). Thus targeting of ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway would be the result of retention and retrieval modulated by the C-terminal phenylalanines.Here we provide evidence that the second determinant for intracellular targeting of ERGIC-53 is constituted by a combination of the lumenal and transmembrane domains rather than by RSQQE. Analysis of ERGIC-53 constructs stably expressed in Lec-1 cells (46Stanley P. Caillibot V. Siminovitch L. Cell. 1975; 6: 121-128Abstract Full Text PDF PubMed Scopus (181) Google Scholar) suggests that this second targeting determinant mediates retention of ERGIC-53 in the ER rather than the ERGIC. Most surprisingly, ER-exit of ERGIC-53 was dependent on the two C-terminal phenylalanines indicating that the cytosolic tail of ERGIC-53 operates as an ERexit determinant that is required for efficient transport of ERGIC-53 to the ERGIC.In vitro binding studies revealed a specific and phenylalanine-dependent interaction between the cytosolic tail of ERGIC-53 and Sec23p of the Sec23p·Sec24p complex of COPII. These results suggest that ERGIC-53 is sorted to COPII vesicles by direct interaction with COPII components in a mechanism similar to receptor-mediated endocytosis or dilysine signal-dependent protein retrieval.DISCUSSIONOur previous suggestion that RSQQE may confer intracellular retention was deduced from the combined observations that (a) inactivation of the dilysine signal by replacing the last 4 amino acids with alanines (T53A7) failed to abolish retention, (b) the lumenal domain and the transmembrane domain alone were not sufficient to retain ERGIC-53/CD4 hybrids, and (c) the construct L53T4RSQQEA3, lacking the dilysine signal and in which ERGIC-53's transmembrane domain was replaced by that of CD4, was largely retained (45Itin C. Schindler R. Hauri H.-P. J. Cell Biol. 1995; 131: 57-67Crossref PubMed Scopus (101) Google Scholar). In light of the findings with the new construct T53A10 which is inefficiently transported, we conclude that the dilysine-independent retention of ERGIC-53 is due to a combined action of the lumenal and transmembrane domains rather than RSQQE. The fact that T4A7displays rapid transport kinetics is further evidence against RSQQE acting as a retention signal. The previously reported retention of L53T4RSQQEA3 remains unexplained but may be a due to a cryptic retention motif recovered by the deletion of the four C-terminal amino acids.Experiments with stably transfected Lec-1 cells strongly suggest that the site of retention provided by the lumenal/transmembrane determinant is the ER. This conclusion is based on a comparison of the GM construct, having a wild-type tail, with the GMAA construct in which the two terminal phenylalanines required for fast intracellular transport were replaced by alanines. Based on the lack of endo-D sensitivity it can be firmly concluded that GMAA is unable to reach the mannosidase I Golgi compartment. The finding that a cytosolic tail peptide terminating in KKAA is a better substrate than KKFF for coatomer binding in vitro raises the possibility that GMAA may leave the ER but would quantitatively be retrieved from the ERGIC and thereby not acquire endo-D sensitivity. The results of the AlF4− experiments, however, rule out such a pre-Golgi recycling route for GMAA. AlF4− blocks anterograde and retrograde transport in the ERGIC (66Plutner H. Davidson H.W. Saraste J. Balch W.E. J. Cell Biol. 1992; 119: 1097-1116Crossref PubMed Scopus (173) Google Scholar, 67Schwaninger R. Plutner H. Bokoch G.M. Balch W.E. J. Cell Biol. 1992; 119: 1077-1096Crossref PubMed Scopus (81) Google Scholar).2 Unlike GM, GMAA was not concentrated in the ERGIC in the presence of AlF4− as assessed by immunofluorescence microscopy and Nycodenz gradient centrifugation. The permanent retention of GMAA in the ER is most likely due to the lumenal/transmembrane retention determinant.The finding of an ER-retention determinant in a dilysine signal-bearing membrane protein is not entirely unexpected. There is evidence that additional ER determinants exist that operate independently of the dilysine retrieval signal (71Nilsson T. Warren G. Curr. Opin. Cell Biol. 1994; 6: 517-521Crossref PubMed Scopus (212) Google Scholar). For example, removal of the dilysine motif from the ER enzyme UDP-glucuronosyltransferase fails to abolish ER retention (68Jackson M.R. Nilsson T. Peterson P.A. J. Cell Biol. 1993; 121: 317-333Crossref PubMed Scopus (312) Google Scholar). It is well known that retention of membrane proteins in the Golgi apparatus is, at least in part, mediated by their transmembrane domain (72Machamer C.E. Curr. Opin. Cell Biol. 1993; 5: 606-612Crossref PubMed Scopus (91) Google Scholar, 73Colley K.J. Glycobiology. 1997; 7: 1-13Crossref PubMed Scopus (283) Google Scholar). The length rather than the amino acid sequence was shown to be important for this localization (74Munro S. EMBO J. 1995; 14: 4695-4704Crossref PubMed Scopus (341) Google Scholar). Likewise, membrane length is important for targeting the C-terminally anchored protein cytochrome b 5 and the t-SNARE Ufe1p to the ER (75Pedrazzini E. Villa A. Borgese N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4207-4212Crossref PubMed Scopus (89) Google Scholar, 76Rayner J.C. Pelham H.R.B. EMBO J. 1997; 16: 1832-1841Crossref PubMed Scopus (150) Google Scholar), although in the latter case the presence of polar amino acids clustered on one side of a helical wheel were also important. Replacement of the transmembrane domain of ERGIC-53 by that of CD4 severely affects the intracellular retention of ERGIC-53 constructs. The transmembrane domain of CD4 has 21 amino acids, hence is 3 amino acids longer than that of ERGIC-53. Further experiments are required to determine if ER retention of ERGIC-53 simply depends on the length of its transmembrane domain or requires specific sequence information.That the cytosolic sequence RSQQEA5FF can overcome the lumenal/transmembrane retention may be due to two fundamentally different mechanisms. Either RSQQEA5FF operates as a transport determinant or it is required for correct folding of ERGIC-53. We consider the latter possibility unlikely because all the constructs we have included in our analysis form disulfide-linked dimers and hexamers with wild-type kinetics. Correct oligomerization is generally considered to reflect correct folding (77Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (775) Google Scholar).Experiments in permanently transfected Lec-1 cells revealed that the two C-terminal phenylalanines are strictly required for ER exit. This strongly suggests that the anterograde targeting determinant in ERGIC-53 mediates exit from the ER. We conclude that the cytosolic sequence RSQQEAAAAAFF is part of an ER-exit determinant which is required for the unique dynamic targeting of ERGIC-53 to the intermediate compartment and cis-Golgi. Consistent with this, ourin vitro binding experiments revealed significant binding of the COP II coat component Sec23p to cytosolic tail of ERGIC-53 and the cytosolic sequence RSQQEAAAAAFF. In yeast and higher eukaryotes Sec23p forms a stable complex with Sec24p under buffer conditions that can be compared with those we have used in our binding assays (10Paccaud J.-P. Reith W. Carpentier J.-L. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: 1535-1546Crossref PubMed Scopus (96) Google Scholar, 78Hicke L. Yoshihisa T. Schekman R. Mol. Biol. Cell. 1992; 3: 667-676Crossref PubMed Scopus (90) Google Scholar). No similar binding was observed for Sec13p or α- and γ-adaptin, components of the plasma membrane and trans-Golgi network-adaptor complexes, respectively, suggesting that the cytosolic tail of ERGIC-53 has a specific affinity for Sec23p·Sec24p subcomplex and does not just bind any kind of cytosolic coat. Interaction with Sec23p was dependent on the two C-terminal phenylalanines that are part of the ER-exit determinant identified by in vivo experiments.We confirm that the cytosolic tail of ERGIC-53 interacts with COPI (79Tisdale E.J. Plutner H. Matteson J. Balch W.E. J. Cell Biol. 1997; 137: 581-593Crossref PubMed Scopus (76) Google Scholar). Interestingly, a peptide with a KKAA dilysine signal showed a much stronger COPI binding than the wild-type peptide bearing the dilysine signal KKFF. The relatively weak COPI binding of the cytosolic tail of ERGIC-53 may explain why ERGIC-53 is not completely recycled from the intermediate compartment but can partly escape to and recycle via the cis-Golgi. To exclude the possibility that the lack of Sec23p binding to the KKAA peptide (i.e. peptide 2) was due to competition by strong COPI interaction, binding assays were also performed with COPI-depleted cell lysate. COPI depletion did not modify the efficiency of Sec23p binding arguing against such a competition.A recent study with cytosolic tail sequences of members of the p24 family of putative cargo receptors suggested a bimodal COPI interaction. Dilysine retrieval signals were reported to bind to the B subcomplex and phenylalanine-dependent forward signals to the F subcomplex (25Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar). At variance with these results, we found no binding of ERGIC-53 to the F subcomplex of COPI under high salt conditions which are necessary to reveal the bimodal COPI binding. In these experiments the cytosolic tail of ERGIC-53 bound to the B subcomplex only and hence behaves like a classical dilysine signal.An antibody directed against the cytosolic tail of ERGIC-53 was recently reported to block the recycling of p58, the rodent homologue of ERGIC-53, in the ERGIC of semi-intact cells (79Tisdale E.J. Plutner H. Matteson J. Balch W.E. J. Cell Biol. 1997; 137: 581-593Crossref PubMed Scopus (76) Google Scholar). The dominant epitope recognized by the antibody includes the C-terminal phenylalanines with weaker contribution of the adjacent lysine residues. The antibody had no effect on ER-exit of p58 which is at variance with our finding that ER-exit is dependent on the two C-terminal phenylalanines. Perhaps the cytosolic tail of ERGIC-53 is masked at the level of the ER and only accessible to the antibody when the protein arrives in the ERGIC (37Lahtinen U. Hellman U. Wernstedt C. Saraste J. Pettersson R.F. J. Biol. Chem. 1996; 271: 4031-4037Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).We believe that direct interaction of Sec23p with the cytoplasmic domain collects ERGIC-53 into COPII vesicles budding from the ER. This notion is in line with the current model of how COPII vesicles bud from the ER (3Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar); ER-bound Sar1p-GTP first recruits the Sec23p·Sec24p complex. The binary complex of Sar1p-GTP and Sec23p·Sec24p can freely diffuse in the plane of the ER membrane, sampling potential cargo proteins by collisional encounter. Favorable interaction with a cargo protein may somehow mark the Sec23p complex for transport. Sec23p-activated cargo may then be clustered and thereby concentrated by multivalent interaction with the Sec13p complex. Accumulation of such coat patches could deform the membrane, creating a bud and ultimately a transport vesicle. ERGIC-53 is perhaps the first cargo protein for which such a sorting mechanism into COPII vesicles might be suggested. In vitro the cytosolic tail of ERGIC-53 interacts specifically with Sec23p in a diphenylalanine-dependent manner, but the cytosolic tail of ERGIC-53 is probably not sufficient to mediate ER-exit in vivo. CD4 chimeras with the cytosolic tail of ERGIC-53 or the cytosolic tail and transmembrane domain of ERGIC-53 are not targeted to post-ER compartments to the same extent as wild-type ERGIC-53 (45Itin C. Schindler R. Hauri H.-P. J. Cell Biol. 1995; 131: 57-67Crossref PubMed Scopus (101) Google Scholar). Similar chimeras with the dimeric reporter protein CD8 and the trimeric reporter VSV-G protein gave identical results, 3F. Kappeler and H.-P. Hauri, unpublished results. indicating that if Sec23p interacts with the cytosolic tail of these ERGIC-53 chimeric proteins this may not be sufficient to mark the Sec23p complex for transport. It is conceivable that the cytosolic tail of ERGIC-53 must be presented in an optimal way, for instance as a hexamer, to successfully induce Sec23p-mediated clustering.In conclusion, we show that the intracellular lectin ERGIC-53 has a cytosolic ER-exit determinant that interacts with the Sec23p complex of COPII. These findings support the notion that anterograde transport from the ER is selective and involves active protein recruitment into budding transport vesicles in a way similar to receptor-mediated endocytosis or COPI-dependent retrieval. In addition the presence of an ER-exit determinant in ERGIC-53 provides further evidence that ERGIC-53 may function as a sorting receptor for glycoproteins in the early secretory pathway. Cycling of ERGIC-53 in the early secretory pathway is controlled by a complex interplay of at least three targeting determinants mediating ER-retention, ER-exit, and retrieval from post-ER compartments and of two types of vesicular coats. The early secretory pathway of higher eukaryotes includes the endoplasmic reticulum (ER), 1The abbreviations used are: ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; endo H, endo-β-N-acetylglucosaminidase H; mAb, monoclonal antibody.1The abbreviations used are: ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; endo H, endo-β-N-acetylglucosaminidase H; mAb, monoclonal antibody.the ER-Golgi intermediate compartment (ERGIC), and the Golgi apparatus that are connected by vesicle-mediated anterograde and retrograde protein transport pathways (1Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 2Bednarek S.Y. Orci L. Schekman R. Trends Cell Biol. 1996; 6: 468-473Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 3Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar, 4Bannykh S.I. Balch W.E. J. Cell Biol. 1997; 138: 1-4Crossref PubMed Scopus (192) Google Scholar, 5Farquhar M.G. Hauri H.-P. Berger E.G. Roth J. The Golgi Apparatus. Birkhäuser Verlag, Basel1997: 63-130Crossref Google Scholar, 6Hauri H.-P. Schweizer A. Hoffman J.F. Jamieson J.D. Handbook of Physiology: Cell Physiology. Oxford University Press for the American Society for Cell Biology, Oxford1997: 605-647Google Scholar, 7Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). Vesicle budding is driven by the recruitment of cytosolic coat proteins to the donor membrane resulting in the formation of a coated vesicle. Two different cytosolic coat complexes, COPI and COPII, are involved in the formation of at least two distinct classes of transport vesicles (3Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar). COPII mediates vesicle budding from the ER for anterograde protein transport (8Barlowe 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, 9Shaywitz D.A. Orci L. Ravazzola M. Swaroop A. Kaiser C.A. J. Cell Biol. 1995; 128: 769-777Crossref PubMed Scopus (85) Google Scholar, 10Paccaud J.-P. Reith W. Carpentier J.-L. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: 1535-1546Crossref PubMed Scopus (96) Google Scholar, 11Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. 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