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Sorting Determinants in the Transmembrane Domain of p24 Proteins

Sorting Determinants in the Transmembrane Domain of p24 Proteins THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 40, Issue of October 3, pp. 24739 –24742, 1997 Communication © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cargo receptors, possibly including p24 proteins (10 –16) and Sorting Determinants in the VIP36-like lectins (17–19), are proposed to move bidirectionally Transmembrane Domain of in the secretory pathway to carry out their function (1, 2). The p24 proteins have previously been identified as components of p24 Proteins* COPI- and COPII-coated vesicles in mammals and yeast, re- spectively (12–14). They form a large family of type I integral (Received for publication, June 27, 1997, and in revised form, membrane proteins with a characteristically short cytoplasmic August 7, 1997) tail, a predicted coiled-coil domain juxtaposed to the TMD in Klaus Fiedler‡ and James E. Rothman the luminal/exoplasmic region, and a highly variable NH - From the Cellular Biochemistry and Biophysics terminal domain (10 –16, 20). Based on the analysis of yeast Program, Memorial Sloan-Kettering Cancer Center, strains lacking the p24 members Emp24p and/or Erv25p, it New York, New York 10021 was suggested that p24 proteins might be involved in the Members of the p24 family of putative cargo receptors packaging of cargo molecules into vesicles as well as vesicle are proposed to contain retrograde and anterograde budding (13–15). Furthermore, yeast strains lacking Emp24p trafficking signals in their cytoplasmic domain to facil- were found to be defective in the retention of ER-resident itate coat protein binding and cycling in the secretory proteins (21) consistent with a role of p24 proteins in control- pathway. We have analyzed the role of the transmem- ling the fidelity of cargo recruitment into budding vesicles. brane domain (TMD) of a p24 protein isolated from We have previously shown that conserved phenylalanine COPI-coated intra-Golgi transport vesicles. CD8-p24 residues in the cytoplasmic domain of p24 proteins mediate the chimeras were transiently expressed in COS7 cells and interaction with coatomer and are likely to be involved in analyzed by immunofluorescence and pulse-chase ex- anterograde trafficking of p24s from the ER through the Golgi periments. The localization and transit of the wild-type complex (12, 20). As a first step toward the identification of chimera from the endoplasmic reticulum (ER) through additional components of the vesicle budding machinery we the Golgi complex involved a glutamic acid residue and have further characterized the transport signals of p24 pro- a conserved glutamine in the TMD. The TMD glutamic teins. A charged amino acid in the p24 TMD appears to serve as acid mediated the localization of the chimeras to the ER a retention signal that is modulated by other TMD residues in the absence of the conserved glutamine. Efficient ER and phenylalanines in the cytoplasmic domain. exit required the TMD glutamine and was further facil- itated by a pair of phenylalanine residues in the cyto- EXPERIMENTAL PROCEDURES plasmic tail. TMD residues of p24 proteins may mediate Materials—The OKT8 CD8 monoclonal antibody was obtained from the interaction with integral membrane proteins of the Ortho Diagnostic Systems (Raritan, NJ), the goat anti-mouse fluores- vesicle budding machinery to ensure p24 packaging into cein-conjugated secondary antibody from Molecular Probes (Eugene, transport vesicles. TM OR), and Lipofectin and LipofectAMINE 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 [ S]methionine/ Transport in the secretory pathway is mediated by a vesic- cysteine from ICN. ular carrier mechanism that allows the cells to preserve or- Construction of CD8 Chimera—The CD8 chimera were constructed ganelle identity by selectively packaging cargo molecules des- by the polymerase chain reaction such that the 165-amino acids of the tined for secretion (1, 2). Trafficking between the ER and the human CD8 extracellular domain were preserved (20). Codon 166 was Golgi apparatus as well as intra-Golgi transport (1– 4) employs changed to glycine to introduce a unique ApaI restriction site, followed coatomer, a complex of seven subunit proteins (5). The COPI by a conserved proline, a stop codon, and a EcoRI site (CD8-C1). Oligo- nucleotides (Gene Link, Thornwood, NY) coding for the COOH-terminal coat is comprised of coatomer and the GTPase ADP-ribosyla- 34 amino acids of chop24a (RVVLWSFFEALVLVAMTLGQIY- tion factor (6, 7). These two cytosolic proteins are sufficient to YLKRFFEVRRVV) and variants thereof (see Fig. 1B), preceded by an pinch off COPI-coated vesicles from Golgi membranes in a ApaI site and followed by a stop codon and EcoRI site, were annealed, cell-free reaction (8). Anterograde- and retrograde-directed subcloned into the CD8 construct, and inserted into the pECE vector COPI-coated vesicles bud from every cisternae of the Golgi (22). Sequences were verified by DNA sequencing using the Sequenase complex in vivo (9). DNA sequencing kit (U. S. Biochemical Corp.). Localization and Pulse-Chase Analysis—Transfection of COS7 cells The sorting of secreted proteins requires adaptor molecules was carried out with Lipofectin and LipofectAMINE for immunofluo- to link cargo to the cytoplasmic vesicle budding machinery. rescence and pulse-chase analysis, respectively, according to the man- ufacturer’s instructions. The chimeras were analyzed 46 h after trans- * This work was supported by the National Institutes of Health (to fection. For localization by immunofluorescence, the cells were J. E. R.) and the Human Frontier Science Program and Swiss National incubated in medium containing 10 mg/ml cycloheximide for 2 h prior to Science Foundation (to K. F.). The costs of publication of this article fixation. They were permeabilized with Triton X-100 before labeling were defrayed in part by the payment of page charges. This article must with the OKT8 CD8 monoclonal antibody (dilution of 1/50) and goat therefore be hereby marked “advertisement” in accordance with 18 anti-mouse fluorescein-conjugated secondary antibody (dilution of U.S.C. Section 1734 solely to indicate this fact. 1/200) as described (17) and viewed and photographed with an Axiophot ‡ To whom correspondence should be addressed: Cellular Biochemis- photomicroscope (Carl Zeiss, Oberkochen, Germany). For pulse-chase try and Biophysics Program, Memorial Sloan-Kettering Cancer Center, analysis COS7 cells were labeled for 20 min with [ S]methionine/ 1275 York Ave., New York, NY 10021. Tel.: 212-639-8445; Fax: cysteine and then incubated for 0, 15, 30, 45, 60, and 120 min in 212-717-3604. medium containing unlabeled methionine/cysteine. The cells were lysed The abbreviations used are: ER, endoplasmic reticulum; CHO, Chi- in Triton X-100, and the CD8 chimeras were isolated by immunopre- nese hamster ovary; TMD, transmembrane domain; v-SNARE, vesicle- cipitation (23) with protein G-agarose before SDS-polyacrylamide gel soluble (N-ethylmaleimide sensitive fusion protein) attachment protein receptor; COP, coat protein. electrophoresis (12% gel) (24). This paper is available on line at http://www.jbc.org 24739 This is an Open Access article under the CC BY license. 24740 Trafficking of p24 Proteins FIG.1. The p24 proteins and CD8-chop24a chimeras. A, the alignment of the COOH-terminal residues of p24 proteins was gener- ated with the GCG program pileup (61) using the available sequences of 16 previously described p24 members (13, 20), the T1/ST2-binding protein (10), and the novel entry hp24g and yp24h. The expressed sequence tags coding for hp24g and yp24h were retrieved by searching the NCBI dbest data base with the program Powerblast using tblastn (available from ftp://ncbi.nlm.nih.gov/pub/sim2/PowerBlast). The prefix a refers to Arabidopsis thaliana, h to human proteins, y to S. cerevisiae, FIG.2. Intracellular localization of CD8-chop24a chimeras by chop24a is from CHO cells (13), gp25l is from dog (11), p23 is from immunofluorescence microscopy. The chimeras were analyzed in rabbit (12), T1/ST2BP is from human, and Emp24p (14) and Erv25p transiently transfected COS7 cells 46 h after transfection. The hybrid (15) are from S. cerevisiae. The amino acid sequences of the TMD and proteins were expressed as EA (A), QA (B), EA-QA (C), QA-FFAA (D), cytoplasmic domain are indicated in single-letter code. The conserved EA-FFAA (E), EA-QA-FFAA (F), and wild-type forms (G) (see Fig. 1B). phenylalanine/hydrophobic residues in the cytoplasmic domain, the The localization of the wild-type and FFAA chimera have been de- conserved glutamine, and the frequently charged/polar residue in the scribed previously (20). Untransfected cells are shown as a control in H. TMD are boxed. GenBank/EMBL accession numbers are as follows. The cells were incubated in medium containing 10 mg/ml cycloheximide ap24a, Z34726; ap24b, T46519; chop24a, U26264; gp25l, X53592; p23, for 2 h prior to fixation. They were permeabilized with Triton X-100 X98303; hp24a, X92098 (16); hp24b, T48838, R25915, T17481; hp24d, before labeling with an antibody to CD8 and a secondary antibody. A set T98284; hp24e, F07445; hp24g, assembled from AA215037 and of representative cells are shown. The exposure time and printing were AA138141; Emp24p, X67317; yp24b, L22015; yp24c, U00059; yp24d, identical for A–H. Bar,15 mm. L22015; yp24e, X87331 and T36996; yp24f, Z48432; Erv25p, Z49810; yp24h, Z72524. B, summary of CD8-chop24a chimeras. CD8 chimeras tion and trafficking, CD8 chimeras were generated in which the were constructed such that the 165 amino acids of the human CD8 CD8 TMD and cytoplasmic domain were replaced with the cor- extracellular domain were preserved, followed by the amino acids gly- cine-proline and the 34 residues of the chop24a TMD and cytoplasmic responding wild-type and mutant sequences of chop24a (Fig. 1B) domain. The wild-type chimera (wt) (20) and mutant chimeras are (13). The CD8 protein has been used previously to analyze cyto- shown. The glutamic acid (position 176 in chop24a), glutamine (position plasmic domain and TMD trafficking signals (25–27). CD8- 187 in chop24a), and phenylalanines (positions 194 and 195 in chop24a) chop24a chimeras were localized by immunofluorescence in tran- that were replaced with alanine in the mutant chimeras are indicated bold underlined. siently expressing COS7 cells (Fig. 2). When the glutamic acid residue alone (EA, panel A), in combination with the conserved RESULTS glutamine (EA-QA, panel C) or phenylalanines (EA-FFAA, panel The p24 family of proteins currently comprises 8 members in E), or all three positions (EA-QA-FFAA, panel F) were replaced mammals and yeast and various orthologues and homologues with alanine, the hybrid proteins were predominantly detected at in other species (Fig. 1A) (10 –16). The alignment of p24 TMDs the cell surface and in a juxtanuclear area, just as the wild-type and cytoplasmic tails showed that in addition to one absolutely chimera had been localized (cf. the previous analysis (20) and conserved phenylalanine in the cytoplasmic domain (position panel G). In contrast, when the glutamine residue alone (QA, 195 in chop24a, the p24 protein isolated from CHO cells (13)), panel B) or in combination with the phenylalanines (QA-FFAA, other amino acids in the TMD are conserved. First, a glutamine panel D) was replaced with alanine, the chimeras were localized residue is present in the TMDs adjacent to the TMD-cytoplas- to the nuclear envelope and tubular-reticular structures, pre- mic domain border in all p24s (position 187 in chop24a). Sec- sumably the ER (Fig. 2). These results suggest that the presence ond, a position juxtaposed to the exoplasmic domain (position of a glutamic acid in the TMD of p24 proteins confers localization 176 in chop24a) accommodates a glutamic acid or polar residue to the early secretory pathway when the conserved TMD gluta- in most p24 proteins with only some exceptions in yeast (Fig. mine residue is absent. 1A). In a helical wheel presentation, the TMD glutamic acid To further corroborate this finding we performed pulse-chase and glutamine (but not the strictly conserved phenylalanine in experiments to measure the rate and extent of transport of the the cytoplasmic domain) line the same side of the predicted chimeras from the ER through the Golgi complex (Fig. 3A). The a-helical structure (data not shown), revealing a relatively time required to receive O-glycans (attached to the CD8 exo- nonhydrophobic face in the TMD of p24 proteins. plasmic domain) that were processed to the mature (sialic To analyze the role of the conserved residues on p24 localiza- acid-containing) form in the medial- or trans-Golgi (28) was Trafficking of p24 Proteins 24741 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. DISCUSSION Sorting signals in the TMD of mammalian and yeast proteins are important for their localization to the Golgi complex and the ER (27, 33– 45), as well as for endocytosis and apical delivery in polarized epithelial cells (46, 47). Recycling of the KDEL receptor and Sec12p from the Golgi to the ER and retention of the coro- navirus E1 protein in the Golgi are likely to require polar resi- dues in the TMD of the proteins (36, 38, 42, 48). For some proteins, trafficking determinants have been found in the TMD as well as in the cytoplasmic domain (a2,6-sialyltransferase, Sed5p, Sec12p, and TGN38) (27, 34, 37, 42). The chop24a mem- ber of the p24 family of putative cargo receptors (13) encodes for a glutamic acid in the TMD. This residue appears to serve as an ER retention signal which is attenuated by a conserved gluta- mine 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 do- main 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 trans- port (20). 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, FIG.3. Pulse-chase analysis of CD8-chop24a chimeras. A, the these observations suggest that the signals present in the TMD mutant chimeras were analyzed in transiently transfected COS7 cells 46 h after transfection. The cells were labeled for 20 min with [ S]me- and cytoplasmic domain modulate each other. The TMD deter- thionine/cysteine and then incubated for 0, 15, 30, 45, 60, and 120 min minant appears to be transplantable since the exchange of the in medium containing unlabeled methionine/cysteine. Then, cells were CD8 TMD alone with the chop24a TMD significantly decreases lysed in Triton X-100, and the CD8 chimeras were isolated by immu- the rate of transport from the ER through the Golgi complex noprecipitation and analyzed by SDS-PAGE. The CD8-chop24a precur- sor (p) and mature (m) O-glycosylated forms (28) are indicated. Immu- (data not shown). noprecipitation from nontransfected cells is shown as a control (Cont.). For a variety of proteins the role of charged amino acids in The wild-type and FFAACD8-chop24a chimeras have been analyzed the TMD has been analyzed in detail. In subunits of the T cell previously (20). B, quantitation of the pulse-chase analysis. The antigen receptor they are involved in quality control of hetero- amounts of the mature O-glycosylated form (28) of each chimera were oligomer assembly by the formation of intramembrane charge determined by densitometric scanning of autoradiograms. The data points for the EA-QA-FFAA and EA-QA CD8-chop24a chimeras repre- pairs (29, 49) and in promoting the formation of intramem- sent average values from two experiments, for the other hybrid proteins brane disulfide-linked dimers (50). Since none of the currently the analysis was carried out once. The CD8-chop24a wild-type and known p24 members contains basic residues or cysteines in the FFAA mutant chimeras (20) are included for comparison. TMD, similar mechanisms for p24 oligomerization seem un- similar for the EA, EA-QA, and EA-FFAA mutant chimeras likely. Nevertheless, the recent analysis of the yeast members and comparable to the wild-type form (Fig. 3B)(cf. Ref. 20). The Emp24p and Erv25p demonstrated that they can be efficiently QA and QA-FFAA mutant hybrid proteins, however, were not cross-linked to each other (15) suggesting that p24 proteins significantly processed within the 2-h chase period (Fig. 3). may form hetero-oligomers. It has not been investigated Charged residues in the TMD confer efficient degradation of whether p24 proteins can also form homo-oligomers. monomeric subunits of the T cell antigen receptor in the ab- Several models could account for the differential localization sence of oligomerization (29 –31). But as expected from the and trafficking of the CD8-chop24a chimeras. It has recently structural context of the chop24a glutamic acid residue (i.e. been shown that similarly charged (glutamic acid) or polar TMD length and positioning) (31, 32), mutant CD8-chop24a (glutamine) TMD residues can promote homodimerization and neu chimera retained in the ER were not rapidly degraded since activation of the receptor tyrosine kinase p185 (51). In the more than 70% of the starting material was present after a 2-h context of p24 proteins this could imply that the TMD is in- chase for all hybrid proteins. volved in oligomerization. If oligomerization of p24 proteins Unexpectedly, the EA-QA-FFAA chimera was processed to the (and CD8-chop24a chimeras) is required for transport from the mature form at a substantially higher rate than all other chime- ER through the Golgi complex, then the TMD alone is appar- ras, including the wild-type CD8-chop24a form (Fig. 3B)(cf. Ref. ently not sufficient for rapid transit. The FFAA chimera in 20). Apparently, the replacement of all three positions allowed which the phenylalanines in the cytoplasmic domain were re- 24742 Trafficking of p24 Proteins 5784 –5789 placed with alanine (Fig. 3 (20)) was transported from the ER 11. Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J., Louvard, D., through the Golgi complex at a significantly lower rate than the Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. (1991) J. Biol. wild-type form. Thus, rapid transit requires the phenylalanine Chem. 266, 19599 –19610 12. Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., residues to aid oligomerization and/or to facilitate interaction Fiedler, K., Helms, J. B., and Wieland, F. T. (1996) J. Cell Biol. 135, with coatomer (20). Although this model could explain the 1239 –1248 13. Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., modulatory interactions among TMD and cytoplasmic tail sig- Tempst, P., and Rothman, J. E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, nals it does not account for the highly efficient transport of the 8011– 8015 EA-QA-FFAA CD8-chop24a chimera. Moreover, this premise 14. Schimmoller, F., Singer-Kruger, B., Schroder, S., Kruger, U., Barlowe, C., and Riezman, H. (1995) EMBO J. 14, 1329 –1339 does not provide an explanation for the apparent decreased 15. Belden, W. J., and Barlowe, C. (1996) J. Biol. Chem. 271, 26939 –26946 rate of transit of CD8-chop24a chimeras that retain only one of 16. Blum, R., Feick, P., Puype, M., Vanderkerckhove, J., Klengel, R., Nastainczyk, the three positions, i.e. the TMD glutamine residue (EA- W., and Schulz, I. (1996) J. Biol. Chem. 271, 17183–17189 17. Fiedler, K., Parton, R. G., Kellner, R., Etzold, T., and Simons, K. (1994) EMBO FFAA), the glutamic acid (QA-FFAA), or the phenylalanine J. 13, 1729 –1740 residues in the cytoplasmic domain (EA-QA). 18. Schroder, S., Schimmoller, F., Singer-Kruger, B., and Riezman, H. (1995) J. Cell Biol. 131, 895–912 Precedence for an alternative model is provided from the 19. Itin, C., Roche, A. C., Monsigny, M., and Hauri, H. P. (1996) Mol. Biol. Cell 7, analysis of Sec12p in yeast. Two glutamines in the Sec12p 483– 493 TMD were shown to be required for the recycling of escaped 20. Fiedler, K., Veit, M., Stamnes, M. A., and Rothman, J. E. (1996) Science 273, 1396 –1399 protein from the Golgi complex back to the ER (42). Proper 21. Elrod-Erickson, M. J., and Kaiser, C. A. (1996) Mol. Biol. Cell 7, 1043–1058 Sec12p ER localization required Rer1p, a protein primarily 22. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A., and Rutter, W. J. localized to the Golgi complex (40 – 42, 52). Rer1p contains four (1986) Cell 45, 721–732 23. Jackson, M. 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Chem. 267, 25196 –25201 tions could ensure the availability of essential machinery com- 29. Cosson, P., Lankford, S. P., Bonifacino, J. S., and Klausner, R. D. (1991) Nature 351, 414 – 416 ponents at the budding site (e.g. v-SNAREs (53)) and might be 30. Bonifacino, J. S., Cosson, P., and Klausner, R. D. (1990) Cell 63, 503–513 mediated by the TMDs of the interacting proteins. Upon inter- 31. Bonifacino, J. S., Cosson, P., Shah, N., and Klausner, R. D. (1991) EMBO J. 10, action with coatomer the p24 proteins would be released into 2783–2793 32. Lankford, S. P., Cosson, P., Bonifacino, J. S., and Klausner, R. D. (1993) the budding vesicle. Alternatively, efficient recruitment might J. Biol. Chem. 268, 4814 – 4820 require the simultaneous interaction of coatomer with both 33. Adams, G. A., and Rose, J. K. (1985) Cell 41, 1007–1015 factors which could either trigger dissociation of p24 proteins 34. Banfield, D. K., Lewis, M. J., Rabouille, C., Warren, G., and Pelham, H. R. B. (1994) J. 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Sorting Determinants in the Transmembrane Domain of p24 Proteins

Fiedler, Klaus; Rothman, James E.
Journal of Biological Chemistry – Oct 1, 1997
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 40, Issue of October 3, pp. 24739 –24742, 1997 Communication © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cargo receptors, possibly including p24 proteins (10 –16) and Sorting Determinants in the VIP36-like lectins (17–19), are proposed to move bidirectionally Transmembrane Domain of in the secretory pathway to carry out their function (1, 2). The p24 proteins have previously been identified as components of p24 Proteins* COPI- and COPII-coated vesicles in mammals and yeast, re- spectively (12–14). They form a large family of type I integral (Received for publication, June 27, 1997, and in revised form, membrane proteins with a characteristically short cytoplasmic August 7, 1997) tail, a predicted coiled-coil domain juxtaposed to the TMD in Klaus Fiedler‡ and James E. Rothman the luminal/exoplasmic region, and a highly variable NH - From the Cellular Biochemistry and Biophysics terminal domain (10 –16, 20). Based on the analysis of yeast Program, Memorial Sloan-Kettering Cancer Center, strains lacking the p24 members Emp24p and/or Erv25p, it New York, New York 10021 was suggested that p24 proteins might be involved in the Members of the p24 family of putative cargo receptors packaging of cargo molecules into vesicles as well as vesicle are proposed to contain retrograde and anterograde budding (13–15). Furthermore, yeast strains lacking Emp24p trafficking signals in their cytoplasmic domain to facil- were found to be defective in the retention of ER-resident itate coat protein binding and cycling in the secretory proteins (21) consistent with a role of p24 proteins in control- pathway. We have analyzed the role of the transmem- ling the fidelity of cargo recruitment into budding vesicles. brane domain (TMD) of a p24 protein isolated from We have previously shown that conserved phenylalanine COPI-coated intra-Golgi transport vesicles. CD8-p24 residues in the cytoplasmic domain of p24 proteins mediate the chimeras were transiently expressed in COS7 cells and interaction with coatomer and are likely to be involved in analyzed by immunofluorescence and pulse-chase ex- anterograde trafficking of p24s from the ER through the Golgi periments. The localization and transit of the wild-type complex (12, 20). As a first step toward the identification of chimera from the endoplasmic reticulum (ER) through additional components of the vesicle budding machinery we the Golgi complex involved a glutamic acid residue and have further characterized the transport signals of p24 pro- a conserved glutamine in the TMD. The TMD glutamic teins. A charged amino acid in the p24 TMD appears to serve as acid mediated the localization of the chimeras to the ER a retention signal that is modulated by other TMD residues in the absence of the conserved glutamine. Efficient ER and phenylalanines in the cytoplasmic domain. exit required the TMD glutamine and was further facil- itated by a pair of phenylalanine residues in the cyto- EXPERIMENTAL PROCEDURES plasmic tail. TMD residues of p24 proteins may mediate Materials—The OKT8 CD8 monoclonal antibody was obtained from the interaction with integral membrane proteins of the Ortho Diagnostic Systems (Raritan, NJ), the goat anti-mouse fluores- vesicle budding machinery to ensure p24 packaging into cein-conjugated secondary antibody from Molecular Probes (Eugene, transport vesicles. TM OR), and Lipofectin and LipofectAMINE 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 [ S]methionine/ Transport in the secretory pathway is mediated by a vesic- cysteine from ICN. ular carrier mechanism that allows the cells to preserve or- Construction of CD8 Chimera—The CD8 chimera were constructed ganelle identity by selectively packaging cargo molecules des- by the polymerase chain reaction such that the 165-amino acids of the tined for secretion (1, 2). Trafficking between the ER and the human CD8 extracellular domain were preserved (20). Codon 166 was Golgi apparatus as well as intra-Golgi transport (1– 4) employs changed to glycine to introduce a unique ApaI restriction site, followed coatomer, a complex of seven subunit proteins (5). The COPI by a conserved proline, a stop codon, and a EcoRI site (CD8-C1). Oligo- nucleotides (Gene Link, Thornwood, NY) coding for the COOH-terminal coat is comprised of coatomer and the GTPase ADP-ribosyla- 34 amino acids of chop24a (RVVLWSFFEALVLVAMTLGQIY- tion factor (6, 7). These two cytosolic proteins are sufficient to YLKRFFEVRRVV) and variants thereof (see Fig. 1B), preceded by an pinch off COPI-coated vesicles from Golgi membranes in a ApaI site and followed by a stop codon and EcoRI site, were annealed, cell-free reaction (8). Anterograde- and retrograde-directed subcloned into the CD8 construct, and inserted into the pECE vector COPI-coated vesicles bud from every cisternae of the Golgi (22). Sequences were verified by DNA sequencing using the Sequenase complex in vivo (9). DNA sequencing kit (U. S. Biochemical Corp.). Localization and Pulse-Chase Analysis—Transfection of COS7 cells The sorting of secreted proteins requires adaptor molecules was carried out with Lipofectin and LipofectAMINE for immunofluo- to link cargo to the cytoplasmic vesicle budding machinery. rescence and pulse-chase analysis, respectively, according to the man- ufacturer’s instructions. The chimeras were analyzed 46 h after trans- * This work was supported by the National Institutes of Health (to fection. For localization by immunofluorescence, the cells were J. E. R.) and the Human Frontier Science Program and Swiss National incubated in medium containing 10 mg/ml cycloheximide for 2 h prior to Science Foundation (to K. F.). The costs of publication of this article fixation. They were permeabilized with Triton X-100 before labeling were defrayed in part by the payment of page charges. This article must with the OKT8 CD8 monoclonal antibody (dilution of 1/50) and goat therefore be hereby marked “advertisement” in accordance with 18 anti-mouse fluorescein-conjugated secondary antibody (dilution of U.S.C. Section 1734 solely to indicate this fact. 1/200) as described (17) and viewed and photographed with an Axiophot ‡ To whom correspondence should be addressed: Cellular Biochemis- photomicroscope (Carl Zeiss, Oberkochen, Germany). For pulse-chase try and Biophysics Program, Memorial Sloan-Kettering Cancer Center, analysis COS7 cells were labeled for 20 min with [ S]methionine/ 1275 York Ave., New York, NY 10021. Tel.: 212-639-8445; Fax: cysteine and then incubated for 0, 15, 30, 45, 60, and 120 min in 212-717-3604. medium containing unlabeled methionine/cysteine. The cells were lysed The abbreviations used are: ER, endoplasmic reticulum; CHO, Chi- in Triton X-100, and the CD8 chimeras were isolated by immunopre- nese hamster ovary; TMD, transmembrane domain; v-SNARE, vesicle- cipitation (23) with protein G-agarose before SDS-polyacrylamide gel soluble (N-ethylmaleimide sensitive fusion protein) attachment protein receptor; COP, coat protein. electrophoresis (12% gel) (24). This paper is available on line at http://www.jbc.org 24739 This is an Open Access article under the CC BY license. 24740 Trafficking of p24 Proteins FIG.1. The p24 proteins and CD8-chop24a chimeras. A, the alignment of the COOH-terminal residues of p24 proteins was gener- ated with the GCG program pileup (61) using the available sequences of 16 previously described p24 members (13, 20), the T1/ST2-binding protein (10), and the novel entry hp24g and yp24h. The expressed sequence tags coding for hp24g and yp24h were retrieved by searching the NCBI dbest data base with the program Powerblast using tblastn (available from ftp://ncbi.nlm.nih.gov/pub/sim2/PowerBlast). The prefix a refers to Arabidopsis thaliana, h to human proteins, y to S. cerevisiae, FIG.2. Intracellular localization of CD8-chop24a chimeras by chop24a is from CHO cells (13), gp25l is from dog (11), p23 is from immunofluorescence microscopy. The chimeras were analyzed in rabbit (12), T1/ST2BP is from human, and Emp24p (14) and Erv25p transiently transfected COS7 cells 46 h after transfection. The hybrid (15) are from S. cerevisiae. The amino acid sequences of the TMD and proteins were expressed as EA (A), QA (B), EA-QA (C), QA-FFAA (D), cytoplasmic domain are indicated in single-letter code. The conserved EA-FFAA (E), EA-QA-FFAA (F), and wild-type forms (G) (see Fig. 1B). phenylalanine/hydrophobic residues in the cytoplasmic domain, the The localization of the wild-type and FFAA chimera have been de- conserved glutamine, and the frequently charged/polar residue in the scribed previously (20). Untransfected cells are shown as a control in H. TMD are boxed. GenBank/EMBL accession numbers are as follows. The cells were incubated in medium containing 10 mg/ml cycloheximide ap24a, Z34726; ap24b, T46519; chop24a, U26264; gp25l, X53592; p23, for 2 h prior to fixation. They were permeabilized with Triton X-100 X98303; hp24a, X92098 (16); hp24b, T48838, R25915, T17481; hp24d, before labeling with an antibody to CD8 and a secondary antibody. A set T98284; hp24e, F07445; hp24g, assembled from AA215037 and of representative cells are shown. The exposure time and printing were AA138141; Emp24p, X67317; yp24b, L22015; yp24c, U00059; yp24d, identical for A–H. Bar,15 mm. L22015; yp24e, X87331 and T36996; yp24f, Z48432; Erv25p, Z49810; yp24h, Z72524. B, summary of CD8-chop24a chimeras. CD8 chimeras tion and trafficking, CD8 chimeras were generated in which the were constructed such that the 165 amino acids of the human CD8 CD8 TMD and cytoplasmic domain were replaced with the cor- extracellular domain were preserved, followed by the amino acids gly- cine-proline and the 34 residues of the chop24a TMD and cytoplasmic responding wild-type and mutant sequences of chop24a (Fig. 1B) domain. The wild-type chimera (wt) (20) and mutant chimeras are (13). The CD8 protein has been used previously to analyze cyto- shown. The glutamic acid (position 176 in chop24a), glutamine (position plasmic domain and TMD trafficking signals (25–27). CD8- 187 in chop24a), and phenylalanines (positions 194 and 195 in chop24a) chop24a chimeras were localized by immunofluorescence in tran- that were replaced with alanine in the mutant chimeras are indicated bold underlined. siently expressing COS7 cells (Fig. 2). When the glutamic acid residue alone (EA, panel A), in combination with the conserved RESULTS glutamine (EA-QA, panel C) or phenylalanines (EA-FFAA, panel The p24 family of proteins currently comprises 8 members in E), or all three positions (EA-QA-FFAA, panel F) were replaced mammals and yeast and various orthologues and homologues with alanine, the hybrid proteins were predominantly detected at in other species (Fig. 1A) (10 –16). The alignment of p24 TMDs the cell surface and in a juxtanuclear area, just as the wild-type and cytoplasmic tails showed that in addition to one absolutely chimera had been localized (cf. the previous analysis (20) and conserved phenylalanine in the cytoplasmic domain (position panel G). In contrast, when the glutamine residue alone (QA, 195 in chop24a, the p24 protein isolated from CHO cells (13)), panel B) or in combination with the phenylalanines (QA-FFAA, other amino acids in the TMD are conserved. First, a glutamine panel D) was replaced with alanine, the chimeras were localized residue is present in the TMDs adjacent to the TMD-cytoplas- to the nuclear envelope and tubular-reticular structures, pre- mic domain border in all p24s (position 187 in chop24a). Sec- sumably the ER (Fig. 2). These results suggest that the presence ond, a position juxtaposed to the exoplasmic domain (position of a glutamic acid in the TMD of p24 proteins confers localization 176 in chop24a) accommodates a glutamic acid or polar residue to the early secretory pathway when the conserved TMD gluta- in most p24 proteins with only some exceptions in yeast (Fig. mine residue is absent. 1A). In a helical wheel presentation, the TMD glutamic acid To further corroborate this finding we performed pulse-chase and glutamine (but not the strictly conserved phenylalanine in experiments to measure the rate and extent of transport of the the cytoplasmic domain) line the same side of the predicted chimeras from the ER through the Golgi complex (Fig. 3A). The a-helical structure (data not shown), revealing a relatively time required to receive O-glycans (attached to the CD8 exo- nonhydrophobic face in the TMD of p24 proteins. plasmic domain) that were processed to the mature (sialic To analyze the role of the conserved residues on p24 localiza- acid-containing) form in the medial- or trans-Golgi (28) was Trafficking of p24 Proteins 24741 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. DISCUSSION Sorting signals in the TMD of mammalian and yeast proteins are important for their localization to the Golgi complex and the ER (27, 33– 45), as well as for endocytosis and apical delivery in polarized epithelial cells (46, 47). Recycling of the KDEL receptor and Sec12p from the Golgi to the ER and retention of the coro- navirus E1 protein in the Golgi are likely to require polar resi- dues in the TMD of the proteins (36, 38, 42, 48). For some proteins, trafficking determinants have been found in the TMD as well as in the cytoplasmic domain (a2,6-sialyltransferase, Sed5p, Sec12p, and TGN38) (27, 34, 37, 42). The chop24a mem- ber of the p24 family of putative cargo receptors (13) encodes for a glutamic acid in the TMD. This residue appears to serve as an ER retention signal which is attenuated by a conserved gluta- mine 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 do- main 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 trans- port (20). 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, FIG.3. Pulse-chase analysis of CD8-chop24a chimeras. A, the these observations suggest that the signals present in the TMD mutant chimeras were analyzed in transiently transfected COS7 cells 46 h after transfection. The cells were labeled for 20 min with [ S]me- and cytoplasmic domain modulate each other. The TMD deter- thionine/cysteine and then incubated for 0, 15, 30, 45, 60, and 120 min minant appears to be transplantable since the exchange of the in medium containing unlabeled methionine/cysteine. Then, cells were CD8 TMD alone with the chop24a TMD significantly decreases lysed in Triton X-100, and the CD8 chimeras were isolated by immu- the rate of transport from the ER through the Golgi complex noprecipitation and analyzed by SDS-PAGE. The CD8-chop24a precur- sor (p) and mature (m) O-glycosylated forms (28) are indicated. Immu- (data not shown). noprecipitation from nontransfected cells is shown as a control (Cont.). For a variety of proteins the role of charged amino acids in The wild-type and FFAACD8-chop24a chimeras have been analyzed the TMD has been analyzed in detail. In subunits of the T cell previously (20). B, quantitation of the pulse-chase analysis. The antigen receptor they are involved in quality control of hetero- amounts of the mature O-glycosylated form (28) of each chimera were oligomer assembly by the formation of intramembrane charge determined by densitometric scanning of autoradiograms. The data points for the EA-QA-FFAA and EA-QA CD8-chop24a chimeras repre- pairs (29, 49) and in promoting the formation of intramem- sent average values from two experiments, for the other hybrid proteins brane disulfide-linked dimers (50). Since none of the currently the analysis was carried out once. The CD8-chop24a wild-type and known p24 members contains basic residues or cysteines in the FFAA mutant chimeras (20) are included for comparison. TMD, similar mechanisms for p24 oligomerization seem un- similar for the EA, EA-QA, and EA-FFAA mutant chimeras likely. Nevertheless, the recent analysis of the yeast members and comparable to the wild-type form (Fig. 3B)(cf. Ref. 20). The Emp24p and Erv25p demonstrated that they can be efficiently QA and QA-FFAA mutant hybrid proteins, however, were not cross-linked to each other (15) suggesting that p24 proteins significantly processed within the 2-h chase period (Fig. 3). may form hetero-oligomers. It has not been investigated Charged residues in the TMD confer efficient degradation of whether p24 proteins can also form homo-oligomers. monomeric subunits of the T cell antigen receptor in the ab- Several models could account for the differential localization sence of oligomerization (29 –31). But as expected from the and trafficking of the CD8-chop24a chimeras. It has recently structural context of the chop24a glutamic acid residue (i.e. been shown that similarly charged (glutamic acid) or polar TMD length and positioning) (31, 32), mutant CD8-chop24a (glutamine) TMD residues can promote homodimerization and neu chimera retained in the ER were not rapidly degraded since activation of the receptor tyrosine kinase p185 (51). In the more than 70% of the starting material was present after a 2-h context of p24 proteins this could imply that the TMD is in- chase for all hybrid proteins. volved in oligomerization. If oligomerization of p24 proteins Unexpectedly, the EA-QA-FFAA chimera was processed to the (and CD8-chop24a chimeras) is required for transport from the mature form at a substantially higher rate than all other chime- ER through the Golgi complex, then the TMD alone is appar- ras, including the wild-type CD8-chop24a form (Fig. 3B)(cf. 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