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• W2048715836 abstract "Members of the p24 family of putative cargo receptors are proposed to contain retrograde and anterograde trafficking signals in their cytoplasmic domain to facilitate coat protein binding and cycling in the secretory pathway. We have analyzed the role of the transmembrane domain (TMD) of a p24 protein isolated from COPI-coated intra-Golgi transport vesicles. CD8-p24 chimeras were transiently expressed in COS7 cells and analyzed by immunofluorescence and pulse-chase experiments. The localization and transit of the wild-type chimera from the endoplasmic reticulum (ER) through the Golgi complex involved a glutamic acid residue and a conserved glutamine in the TMD. The TMD glutamic acid mediated the localization of the chimeras to the ER in the absence of the conserved glutamine. Efficient ER exit required the TMD glutamine and was further facilitated by a pair of phenylalanine residues in the cytoplasmic tail. TMD residues of p24 proteins may mediate the interaction with integral membrane proteins of the vesicle budding machinery to ensure p24 packaging into transport vesicles. Members of the p24 family of putative cargo receptors are proposed to contain retrograde and anterograde trafficking signals in their cytoplasmic domain to facilitate coat protein binding and cycling in the secretory pathway. We have analyzed the role of the transmembrane domain (TMD) of a p24 protein isolated from COPI-coated intra-Golgi transport vesicles. CD8-p24 chimeras were transiently expressed in COS7 cells and analyzed by immunofluorescence and pulse-chase experiments. The localization and transit of the wild-type chimera from the endoplasmic reticulum (ER) through the Golgi complex involved a glutamic acid residue and a conserved glutamine in the TMD. The TMD glutamic acid mediated the localization of the chimeras to the ER in the absence of the conserved glutamine. Efficient ER exit required the TMD glutamine and was further facilitated by a pair of phenylalanine residues in the cytoplasmic tail. TMD residues of p24 proteins may mediate the interaction with integral membrane proteins of the vesicle budding machinery to ensure p24 packaging into transport vesicles. Transport in the secretory pathway is mediated by a vesicular carrier mechanism that allows the cells to preserve organelle identity by selectively packaging cargo molecules destined for secretion (1Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar, 2Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (819) Google Scholar). Trafficking between the ER 1The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; TMD, transmembrane domain; v-SNARE, vesicle-soluble (N-ethylmaleimide sensitive fusion protein) attachment protein receptor; COP, coat protein.and the Golgi apparatus as well as intra-Golgi transport (1Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar, 2Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (819) Google Scholar, 3Kreis T.E. Lowe M. Pepperkok R. Annu. Rev. Cell Dev. Biol. 1995; 11: 677-706Crossref PubMed Scopus (101) Google Scholar, 4Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (81) Google Scholar) employs coatomer, a complex of seven subunit proteins (5Waters M.G. Serafini T. Rothman J.E. Nature. 1991; 349: 248-251Crossref PubMed Scopus (381) Google Scholar). The COPI coat is comprised of coatomer and the GTPase ADP-ribosylation factor (6Donaldson J.G. Kahn R.A. Lippincott S.J. Klausner R.D. Science. 1991; 254: 1197-1199Crossref PubMed Scopus (260) Google Scholar, 7Serafini T. Orci L. Amherdt M. Brunner M. Kahn R.A. Rothman J.E. Cell. 1991; 67: 239-253Abstract Full Text PDF PubMed Scopus (452) Google Scholar). These two cytosolic proteins are sufficient to pinch off COPI-coated vesicles from Golgi membranes in a cell-free reaction (8Orci L. Palmer D.J. Amherdt M. Rothman J.E. Nature. 1993; 364: 732-734Crossref PubMed Scopus (182) Google Scholar). Anterograde- and retrograde-directed COPI-coated vesicles bud from every cisternae of the Golgi complex in vivo (9Orci 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 (352) Google Scholar). The sorting of secreted proteins requires adaptor molecules to link cargo to the cytoplasmic vesicle budding machinery. Cargo receptors, possibly including p24 proteins (10Gayle M.A. Slack J.L. Bonnert T.P. Renshaw B.R. Sonoda G. Taguchi T. Testa J.R. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5784-5789Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Wada I. Rindress D. Cameron P.H. Ou W.J. Doherty J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar, 12Sohn 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 (181) Google Scholar, 13Stamnes 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 (196) Google Scholar, 14Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar, 15Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 16Blum R. Feick P. Puype M. Vanderkerckhove J. Klengel R. Nastainczyk W. Schulz I. J. Biol. Chem. 1996; 271: 17183-17189Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and VIP36-like lectins (17Fiedler K. Parton R.G. Kellner R. Etzold T. Simons K. EMBO J. 1994; 13: 1729-1740Crossref PubMed Scopus (193) Google Scholar, 18Schroder S. Schimmoller F. Singer-Kruger B. Riezman H. J. Cell Biol. 1995; 131: 895-912Crossref PubMed Scopus (157) Google Scholar, 19Itin C. Roche A.C. Monsigny M. Hauri H.P. Mol. Biol. Cell. 1996; 7: 483-493Crossref PubMed Scopus (155) Google Scholar), are proposed to move bidirectionally in the secretory pathway to carry out their function (1Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar, 2Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (819) Google Scholar). The p24 proteins have previously been identified as components of COPI- and COPII-coated vesicles in mammals and yeast, respectively (12Sohn 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 (181) Google Scholar, 13Stamnes 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 (196) Google Scholar, 14Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar). They form a large family of type I integral membrane proteins with a characteristically short cytoplasmic tail, a predicted coiled-coil domain juxtaposed to the TMD in the luminal/exoplasmic region, and a highly variable NH2-terminal domain (10Gayle M.A. Slack J.L. Bonnert T.P. Renshaw B.R. Sonoda G. Taguchi T. Testa J.R. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5784-5789Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Wada I. Rindress D. Cameron P.H. Ou W.J. Doherty J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar, 12Sohn 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 (181) Google Scholar, 13Stamnes 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 (196) Google Scholar, 14Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar, 15Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 16Blum R. Feick P. Puype M. Vanderkerckhove J. Klengel R. Nastainczyk W. Schulz I. J. Biol. Chem. 1996; 271: 17183-17189Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). Based on the analysis of yeast strains lacking the p24 members Emp24p and/or Erv25p, it was suggested that p24 proteins might be involved in the packaging of cargo molecules into vesicles as well as vesicle budding (13Stamnes 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 (196) Google Scholar, 14Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar, 15Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Furthermore, yeast strains lacking Emp24p were found to be defective in the retention of ER-resident proteins (21Elrod-Erickson M.J. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1043-1058Crossref PubMed Scopus (143) Google Scholar) consistent with a role of p24 proteins in controlling the fidelity of cargo recruitment into budding vesicles. We have previously shown that conserved phenylalanine residues in the cytoplasmic domain of p24 proteins mediate the interaction with coatomer and are likely to be involved in anterograde trafficking of p24s from the ER through the Golgi complex (12Sohn 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 (181) Google Scholar, 20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). As a first step toward the identification of additional components of the vesicle budding machinery we have further characterized the transport signals of p24 proteins. A charged amino acid in the p24 TMD appears to serve as a retention signal that is modulated by other TMD residues and phenylalanines in the cytoplasmic domain. The OKT8 CD8 monoclonal antibody was obtained from Ortho Diagnostic Systems (Raritan, NJ), the goat anti-mouse fluorescein-conjugated secondary antibody from Molecular Probes (Eugene, OR), and Lipofectin and LipofectAMINETM from Life Technologies, Inc. COS7 (SV40-transformed African green monkey kidney) cells were purchased from the American Type Culture Collection (Rockville, MD), protein G-agarose from Boehringer Mannheim, and [35S]methionine/cysteine from ICN. The CD8 chimera were constructed by the polymerase chain reaction such that the 165-amino acids of the human CD8 extracellular domain were preserved (20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). Codon 166 was changed to glycine to introduce a unique ApaI restriction site, followed by a conserved proline, a stop codon, and aEcoRI site (CD8-C1). Oligonucleotides (Gene Link, Thornwood, NY) coding for the COOH-terminal 34 amino acids of chop24a (RVVLWSFFEALVLVAMTLGQIYYLKRFFEVRRVV) and variants thereof (see Fig. 1 B), preceded by an ApaI site and followed by a stop codon and EcoRI site, were annealed, subcloned into the CD8 construct, and inserted into the pECE vector (22Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (697) Google Scholar). Sequences were verified by DNA sequencing using the Sequenase DNA sequencing kit (U. S. Biochemical Corp.). Transfection of COS7 cells was carried out with Lipofectin and LipofectAMINE for immunofluorescence and pulse-chase analysis, respectively, according to the manufacturer's instructions. The chimeras were analyzed 46 h after transfection. For localization by immunofluorescence, the cells were incubated in medium containing 10 μg/ml cycloheximide for 2 h prior to fixation. They were permeabilized with Triton X-100 before labeling with the OKT8 CD8 monoclonal antibody (dilution of 1/50) and goat anti-mouse fluorescein-conjugated secondary antibody (dilution of 1/200) as described (17Fiedler K. Parton R.G. Kellner R. Etzold T. Simons K. EMBO J. 1994; 13: 1729-1740Crossref PubMed Scopus (193) Google Scholar) and viewed and photographed with an Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany). For pulse-chase analysis COS7 cells were labeled for 20 min with [35S]methionine/cysteine and then incubated for 0, 15, 30, 45, 60, and 120 min in medium containing unlabeled methionine/cysteine. The cells were lysed in Triton X-100, and the CD8 chimeras were isolated by immunoprecipitation (23Jackson M.R. Nilsson T. Peterson P.A. J. Cell Biol. 1993; 121: 317-333Crossref PubMed Scopus (314) Google Scholar) with protein G-agarose before SDS-polyacrylamide gel electrophoresis (12% gel) (24Hara-Kuge S. Kuge O. Orci L. Amherdt M. Ravazzola M. Wieland F.T. Rothman J.E. J. Cell Biol. 1994; 124: 883-892Crossref PubMed Scopus (117) Google Scholar). The p24 family of proteins currently comprises 8 members in mammals and yeast and various orthologues and homologues in other species (Fig. 1 A) (10Gayle M.A. Slack J.L. Bonnert T.P. Renshaw B.R. Sonoda G. Taguchi T. Testa J.R. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5784-5789Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Wada I. Rindress D. Cameron P.H. Ou W.J. Doherty J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar, 12Sohn 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 (181) Google Scholar, 13Stamnes 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 (196) Google Scholar, 14Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar, 15Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 16Blum R. Feick P. Puype M. Vanderkerckhove J. Klengel R. Nastainczyk W. Schulz I. J. Biol. Chem. 1996; 271: 17183-17189Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The alignment of p24 TMDs and cytoplasmic tails showed that in addition to one absolutely conserved phenylalanine in the cytoplasmic domain (position 195 in chop24a, the p24 protein isolated from CHO cells (13Stamnes 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 (196) Google Scholar)), other amino acids in the TMD are conserved. First, a glutamine residue is present in the TMDs adjacent to the TMD-cytoplasmic domain border in all p24s (position 187 in chop24a). Second, a position juxtaposed to the exoplasmic domain (position 176 in chop24a) accommodates a glutamic acid or polar residue in most p24 proteins with only some exceptions in yeast (Fig. 1 A). In a helical wheel presentation, the TMD glutamic acid and glutamine (but not the strictly conserved phenylalanine in the cytoplasmic domain) line the same side of the predicted α-helical structure (data not shown), revealing a relatively nonhydrophobic face in the TMD of p24 proteins. To analyze the role of the conserved residues on p24 localization and trafficking, CD8 chimeras were generated in which the CD8 TMD and cytoplasmic domain were replaced with the corresponding wild-type and mutant sequences of chop24a (Fig. 1 B) (13Stamnes 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 (196) Google Scholar). The CD8 protein has been used previously to analyze cytoplasmic domain and TMD trafficking signals (25Nilsson T.M. Jackson M.R. Peterson P.A. Cell. 1989; 58: 707-718Abstract Full Text PDF PubMed Scopus (369) Google Scholar, 26Jackson M.R. Nilsson T. Peterson P.A. EMBO J. 1990; 9: 3153-3162Crossref PubMed Scopus (728) Google Scholar, 27Ponnambalam S. Rabouille C. Luzio J.P. Nilsson T. Warren G. J. Cell Biol. 1994; 125: 253-268Crossref PubMed Scopus (120) Google Scholar). CD8-chop24a chimeras were localized by immunofluorescence in transiently expressing COS7 cells (Fig.2). When the glutamic acid residue alone (EA, panel A), in combination with the conserved glutamine (EA-QA, panel C) or phenylalanines (EA-FFAA, panel E), or all three positions (EA-QA-FFAA, panel F) were replaced with alanine, the hybrid proteins were predominantly detected at the cell surface and in a juxtanuclear area, just as the wild-type chimera had been localized (cf. the previous analysis (20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar) and panel G). In contrast, when the glutamine residue alone (QA,panel B) or in combination with the phenylalanines (QA-FFAA, panel D) was replaced with alanine, the chimeras were localized to the nuclear envelope and tubular-reticular structures, presumably the ER (Fig. 2). These results suggest that the presence of a glutamic acid in the TMD of p24 proteins confers localization to the early secretory pathway when the conserved TMD glutamine residue is absent. To further corroborate this finding we performed pulse-chase experiments to measure the rate and extent of transport of the chimeras from the ER through the Golgi complex (Fig.3 A). The time required to receive O-glycans (attached to the CD8 exoplasmic domain) that were processed to the mature (sialic acid-containing) form in the medial- or trans-Golgi (28Pascale M.C. Erra M.C. Malagolini N. Serafini-Cessi F. Leone A. Bonatti S. J. Biol. Chem. 1992; 267: 25196-25201Abstract Full Text PDF PubMed Google Scholar) was similar for the EA, EA-QA, and EA-FFAA mutant chimeras and comparable to the wild-type form (Fig.3 B) (cf. Ref. 20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). The QA and QA-FFAA mutant hybrid proteins, however, were not significantly processed within the 2-h chase period (Fig. 3). Charged residues in the TMD confer efficient degradation of monomeric subunits of the T cell antigen receptor in the absence of oligomerization (29Cosson P. Lankford S.P. Bonifacino J.S. Klausner R.D. Nature. 1991; 351: 414-416Crossref PubMed Scopus (208) Google Scholar, 30Bonifacino J.S. Cosson P. Klausner R.D. Cell. 1990; 63: 503-513Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 31Bonifacino J.S. Cosson P. Shah N. Klausner R.D. EMBO J. 1991; 10: 2783-2793Crossref PubMed Scopus (163) Google Scholar). But as expected from the structural context of the chop24a glutamic acid residue (i.e. TMD length and positioning) (31Bonifacino J.S. Cosson P. Shah N. Klausner R.D. EMBO J. 1991; 10: 2783-2793Crossref PubMed Scopus (163) Google Scholar, 32Lankford S.P. Cosson P. Bonifacino J.S. Klausner R.D. J. Biol. Chem. 1993; 268: 4814-4820Abstract Full Text PDF PubMed Google Scholar), mutant CD8-chop24a chimera retained in the ER were not rapidly degraded since more than 70% of the starting material was present after a 2-h chase for all hybrid proteins. Unexpectedly, the EA-QA-FFAA chimera was processed to the mature form at a substantially higher rate than all other chimeras, including the wild-type CD8-chop24a form (Fig. 3 B) (cf. Ref. 20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). Apparently, the replacement of all three positions allowed efficient transit from the ER through the Golgi complex. Next, we tested whether the chop24a TMD alone alters the trafficking of the reporter molecule CD8 by replacing the CD8 TMD with the respective sequence of chop24a (data not shown). Pulse-chase experiments showed that the rate of transport of the CD8-chop24a chimera was decreased relative to the CD8 wild-type protein. Sorting signals in the TMD of mammalian and yeast proteins are important for their localization to the Golgi complex and the ER (27Ponnambalam S. Rabouille C. Luzio J.P. Nilsson T. Warren G. J. Cell Biol. 1994; 125: 253-268Crossref PubMed Scopus (120) Google Scholar, 33Adams G.A. Rose J.K. Cell. 1985; 41: 1007-1015Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 34Banfield D.K. Lewis M.J. Rabouille C. Warren G. Pelham H.R.B. J. Cell Biol. 1994; 127: 357-371Crossref PubMed Scopus (147) Google Scholar, 35Nilsson T. Lucocq J.M. Mackay D. Warren G. EMBO J. 1991; 10: 3567-3575Crossref PubMed Scopus (160) Google Scholar, 36Swift A.M. Machamer C.E. J. 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Chem. 1997; 272: 1970-1975Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), as well as for endocytosis and apical delivery in polarized epithelial cells (46Lazarovits J. Naim H.Y. Rodriguez A.C. Wang R.H. Fire E. Bird C. Henis Y.I. Roth M.G. J. Cell Biol. 1996; 134: 339-348Crossref PubMed Scopus (22) Google Scholar, 47Kundu A. Avalos R.T. Sanderson C.M. Nayak D.P. J. Virol. 1996; 70: 6508-6515Crossref PubMed Google Scholar). Recycling of the KDEL receptor and Sec12p from the Golgi to the ER and retention of the coronavirus E1 protein in the Golgi are likely to require polar residues in the TMD of the proteins (36Swift A.M. Machamer C.E. J. Cell Biol. 1991; 115: 19-30Crossref PubMed Scopus (136) Google Scholar, 38Townsley F.M. Wilson D.W. Pelham H.R. EMBO J. 1993; 12: 2821-2829Crossref PubMed Scopus (111) Google Scholar, 42Sato M. Sato K. Nakano A. J. Cell Biol. 1996; 134: 279-293Crossref PubMed Scopus (130) Google Scholar, 48Machamer C.E. Grim M.G. Esquela A. Chung S.W. Rolls M. Ryan K. Swift A.M. Mol. Biol. Cell. 1993; 4: 695-704Crossref PubMed Scopus (54) Google Scholar). For some proteins, trafficking determinants have been found in the TMD as well as in the cytoplasmic domain (α2,6-sialyltransferase, Sed5p, Sec12p, and TGN38) (27Ponnambalam S. Rabouille C. Luzio J.P. Nilsson T. Warren G. J. Cell Biol. 1994; 125: 253-268Crossref PubMed Scopus (120) Google Scholar, 34Banfield D.K. Lewis M.J. Rabouille C. Warren G. Pelham H.R.B. J. Cell Biol. 1994; 127: 357-371Crossref PubMed Scopus (147) Google Scholar,37Munro S. EMBO J. 1991; 10: 3577-3588Crossref PubMed Scopus (218) Google Scholar, 42Sato M. Sato K. Nakano A. J. Cell Biol. 1996; 134: 279-293Crossref PubMed Scopus (130) Google Scholar). The chop24a member of the p24 family of putative cargo receptors (13Stamnes 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 (196) Google Scholar) encodes for a glutamic acid in the TMD. This residue appears to serve as an ER retention signal which is attenuated by a conserved glutamine in the TMD and phenylalanine residues in the cytoplasmic tail (Fig. 2). The presence of all three motifs facilitates efficient ER exit and transit through the Golgi complex. Unexpectedly, a EA-QA-FFAA CD8-chop24a hybrid protein that is devoid of all analyzed sorting determinants was delivered to the medial-/trans-Golgi at a significantly higher rate than the wild-type chimera and other hybrid proteins (Fig. 3). Apparently, the presence of phenylalanine residues in the cytoplasmic domain decreases the rate of transit from the ER through the Golgi complex when the TMD glutamic acid and glutamine are absent. Conversely, when the TMD glutamic acid and glutamine are present, the phenylalanine residues increase the rate of transport (20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). Moreover, the presence of the TMD glutamine residue decreases the rate of transport when the TMD glutamic acid and the phenylalanines are replaced with alanine. Taken together, these observations suggest that the signals present in the TMD and cytoplasmic domain modulate each other. The TMD determinant appears to be transplantable since the exchange of the CD8 TMD alone with the chop24a TMD significantly decreases the rate of transport from the ER through the Golgi complex (data not shown). For a variety of proteins the role of charged amino acids in the TMD has been analyzed in detail. In subunits of the T cell antigen receptor they are involved in quality control of hetero-oligomer assembly by the formation of intramembrane charge pairs (29Cosson P. Lankford S.P. Bonifacino J.S. Klausner R.D. Nature. 1991; 351: 414-416Crossref PubMed Scopus (208) Google Scholar, 49Manolios N. Bonifacino J.S. Klausner R.D. Science. 1990; 249: 274-277Crossref PubMed Scopus (192) Google Scholar) and in promoting the formation of intramembrane disulfide-linked dimers (50Rutledge T. Cosson P. Manolios N. Bonifacino J.S. Klausner R.D. EMBO J. 1992; 11: 3245-3254Crossref PubMed Scopus (87) Google Scholar). Since none of the currently known p24 members contains basic residues or cysteines in the TMD, similar mechanisms for p24 oligomerization seem unlikely. Nevertheless, the recent analysis of the yeast members Emp24p and Erv25p demonstrated that they can be efficiently cross-linked to each other (15Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) suggesting that p24 proteins may form hetero-oligomers. It has not been investigated whether p24 proteins can also form homo-oligomers. Several models could account for the differential localization and trafficking of the CD8-chop24a chimeras. It has recently been shown that similarly charged (glutamic acid) or polar (glutamine) TMD residues can promote homodimerization and activation of the receptor tyrosine kinase p185neu (51Chen L.I. Webster M.K. Meyer A.N. Donoghue D.J. J. Cell Biol. 1997; 137: 619-631Crossref PubMed Scopus (41) Google Scholar). In the context of p24 proteins this could imply that the TMD is involved in oligomerization. If oligomerization of p24 proteins (and CD8-chop24a chimeras) is required for transport from the ER through the Golgi complex, then the TMD alone is apparently not sufficient for rapid transit. The FFAA chimera in which the phenylalanines in the cytoplasmic domain were replaced with alanine (Fig. 3 (20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar)) was transported from the ER through the Golgi complex at a significantly lower rate than the wild-type form. Thus, rapid transit requires the phenylalanine residues to aid oligomerization and/or to facilitate interaction with coatomer (20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). Although this model could explain the modulatory interactions among TMD and cytoplasmic tail signals it does not account for the highly efficient transport of the EA-QA-FFAA CD8-chop24a chimera. Moreover, this premise does not provide an explanation for the apparent decreased rate of transit of CD8-chop24a chimeras that retain only one of the three positions, i.e. the TMD glutamine residue (EA-FFAA), the glutamic acid (QA-FFAA), or the phenylalanine residues in the cytoplasmic domain (EA-QA). Precedence for an alternative model is provided from the analysis of Sec12p in yeast. Two glutamines in the Sec12p TMD were shown to be required for the recycling of escaped protein from the Golgi complex back to the ER (42Sato M. Sato K. Nakano A. J. Cell Biol. 1996; 134: 279-293Crossref PubMed Scopus (130) Google Scholar). Proper Sec12p ER localization required Rer1p, a protein primarily localized to the Golgi complex (40Sato K. Nishikawa S. Nakano A. Mol. Biol. Cell. 1995; 6: 1459-1477Crossref PubMed Scopus (92) Google Scholar, 41Boehm J. Ulrich H.D. Ossig R. Schmitt H.D. EMBO J. 1994; 13: 3696-3710Crossref PubMed Scopus (51) Google Scholar, 42Sato M. Sato K. Nakano A. J. Cell Biol. 1996; 134: 279-293Crossref PubMed Scopus (130) Google Scholar, 52Boehm J. Letourneur F. Ballensiefen W. Ossipov D. Demolliere C. Schmitt H.D. J. Cell Sci. 1997; 110: 991-1003Crossref PubMed Google Scholar). Rer1p contains four TMDs that encode for polar as well as charged residues and is proposed to function as a TMD sorting receptor. A direct interaction with Sec12p remains to be shown. Analogous to Sec12p and Rer1p, p24 recruitment could involve not only the interaction with coatomer (20Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar) but also the binding to other integral membrane proteins of the vesicle budding machinery. These additional protein-protein interactions could ensure the availability of essential machinery components at the budding site (e.g.v-SNAREs (53Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar)) and might be mediated by the TMDs of the interacting proteins. Upon interaction with coatomer the p24 proteins would be released into the budding vesicle. Alternatively, efficient recruitment might require the simultaneous interaction of coatomer with both factors which could either trigger dissociation of p24 proteins for recruitment or lead to the recruitment of both components into the budding vesicle. With respect to our analysis of the CD8-chop24a chimeras the second model predicts that the absence of any trafficking signal in the TMD and a coatomer binding motif (e.g. phenylalanines or basic residues) would allow unregulated, passive inclusion into vesicles not restricted by the limited number of coatomer binding sites. This could explain the more rapid transport of the EA-QA-FFAAversus the EA-QA form of the CD8-chop24a chimeras. Similarly, the absence of a TMD signal in a p24 protein (EA-QA-FFAA chimera versus FFAA chimera) could abolish the interaction with a negative regulatory component and effectively increase the rate of transport by bulk flow. It is possible that hitherto unidentified factors, or members of a growing class of multispanning membrane proteins (54Ljungdahl P.O. Gimeno C.J. Styles C.A. Fink G.R. Cell. 1992; 71: 463-478Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 55Kuehn M.J. Schekman R. Ljungdahl P.O. J. Cell Biol. 1996; 135: 585-595Crossref PubMed Scopus (94) Google Scholar, 56Poster J.B. Dean N. J. Biol. Chem. 1996; 271: 3837-3845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 57Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar, 58Stenius K. Janz R. Sudhof T.C. Jahn R. J. Cell Biol. 1995; 131: 1801-1809Crossref PubMed Scopus (73) Google Scholar, 59Zacchetti D. Peranen J. Murata M. Fiedler K. Simons K. FEBS Lett. 1995; 377: 465-469Crossref PubMed Scopus (95) Google Scholar, 60Kim T. Fiedler K. Madison D.L. Krueger W.H. Pfeiffer S.E. J. Neurosci. Res. 1995; 42: 413-422Crossref PubMed Scopus (115) Google Scholar), are involved in the regulation of p24 packaging into budding vesicles. We thank M. Veit, C. Hughes, and F. Parlati for critical comments on the manuscript and for discussions. We are grateful to S. Ponnambalam and T. Nilsson for the CD8 cDNA and to M. Spiess for the pECE vector." @default.
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• W2048715836 title "Sorting Determinants in the Transmembrane Domain of p24 Proteins" @default.
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