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• W2166656822 abstract "After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1282, a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented, N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1282 had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1282 appended to α2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1282, and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins. After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1282, a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented, N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1282 had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1282 appended to α2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1282, and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins. The mechanism(s) by which ER 1The abbreviations used are: ER, endoplasmic reticulum; TM, transmembrane; GPI, glycosylphosphatidylinositol; GPIT, GPI transamidase; TfR, transferrin receptor; Endo H, endoglycosidase H; PNGase F, peptide:N-Glycosidase F; aa, amino acids. -resident proteins are sorted from those destined for post-ER compartments is a subject of ongoing debate (1Teasdale R.D. Jackson M.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 27-54Crossref PubMed Scopus (447) Google Scholar, 2Fewell S.W. Travers K.J. Weissman J.S. Brodsky J.L. Annu. Rev. Genet. 2001; 35: 149-191Crossref PubMed Scopus (264) Google Scholar, 3Barlowe C. Trends Cell Biol. 2003; 13: 295-300Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 4Lee M.C.S. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (712) Google Scholar, 5Otte S. Barlowe C. Nat. Cell Biol. 2004; 6: 1189-1194Crossref PubMed Scopus (82) Google Scholar). Possible scenarios to explain ER retention of proteins include trapping of ER residents within a matrix or oligomeric structure, signal-mediated retention, lack of an export signal, and signal-mediated retrieval from post-ER compartments. These scenarios must be viewed in the context of bulk anterograde membrane flow from the ER that has been hypothesized to nonselectively carry membrane and fluid content to post-ER compartments (6Wieland F.T. Gleason M.L. Serafini T.A. Rothman J.E. Cell. 1987; 50: 289-300Abstract Full Text PDF PubMed Scopus (358) Google Scholar, 7Griffiths G. Doms R.W. Mayhew T. Lucocq J. Trends Cell Biol. 1995; 5: 9-13Abstract Full Text PDF PubMed Scopus (21) Google Scholar). The two best characterized ER localization signals, the KDEL and dilysine motifs, are based on retrieval mechanisms that involve direct or indirect binding of the motif to the COPI coats of vesicles mediating retrograde transport from the Golgi to the ER. The KDEL motif is found on soluble proteins that reside within the ER lumen (8Munro S. Pelham H.R.B. Cell. 1987; 48: 899-907Abstract Full Text PDF PubMed Scopus (1590) Google Scholar, 9Lewis M.J. Pelham H.R. Nature. 1990; 348: 162-163Crossref PubMed Scopus (247) Google Scholar, 10Lewis M.J. Pelham H.R. Cell. 1992; 68: 353-364Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 11Tang B.L. Wong S.H. Qi X.L. Low S.H. Hong W. J. Cell Biol. 1993; 120: 325-328Crossref PubMed Scopus (117) Google Scholar); the cytoplasmically exposed dilysine motif serves a similar function for ER-resident membrane proteins (12Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (484) Google Scholar, 13Letourneur F. Gaynor E.C. Hennecke S. Demolliere C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (674) Google Scholar). However, only a small fraction of any given type of molecule bearing a KDEL or dilysine motif appears to exit the ER and needs to be retrieved; the majority appear not to leave the ER at all. The question remains. How do ER resident proteins remain resident in the ER? It is possible that many ER resident proteins are components of hetero- or homo-oligomeric complexes that in turn interact with other complexes to yield an aggregate that is too large to export efficiently. Consistent with this are observations of the slow lateral diffusion of oligosaccharyltransferase complexes that associate with the protein translocon (14Nikonov A.V. Snapp E. Lippincott-Schwartz J. Kreibich G. J. Cell Biol. 2002; 158: 497-506Crossref PubMed Scopus (53) Google Scholar). Alternatively, some ER residents, like ER chaperones, may be continuously engaged with protein traffic entering the ER such that their own exit is impeded. These interactions or the numerous quasi-interactions that are probably promoted by the high protein density in the ER may be sufficient to retain proteins if the rate of bulk flow of membrane and fluid departing the ER is low and if the proteins lack explicit export signals. We classify these possibilities under the heading of “signalless” retention mechanisms. In contrast, it is possible to consider retention resulting from an explicit signal within the retained protein. For the particular case of ER membrane proteins, it is conceivable that sequence motifs in one or more of the topological domains of the protein (cytoplasmic, transmembrane (TM), or luminal) interact specifically with effectors to block exit of the protein from the ER. However, many ER membrane proteins appear to enjoy relatively unencumbered lateral diffusion in the membrane, suggesting that their presumed exclusion from ER-derived transport vesicles is not due to immobility in the membrane resulting from, for example, cytoskeletal association or large complex formation (15Pentcheva T. Spiliotis E.T. Edidin M. J. Immunol. 2002; 168: 1538-1541Crossref PubMed Scopus (26) Google Scholar, 16Szczesna-Skorupa E. Chen C.D. Rogers S. Kemper B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14793-14798Crossref PubMed Scopus (56) Google Scholar). The few experimentally characterized ER retention motifs, such as cytoplasmic N-terminal and internal diarginine sequences, cytoplasmic C-terminal tyrosine-based sequences, or transmembrane domain sequences, are not universal and/or share no sequence similarity between other proteins (1Teasdale R.D. Jackson M.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 27-54Crossref PubMed Scopus (447) Google Scholar, 17Schutze M.P. Peterson P.A. Jackson M.R. EMBO J. 1994; 13: 1696-1705Crossref PubMed Scopus (269) Google Scholar, 18Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar, 19Mallabiabarrena A. Jimenez M.A. Rico M. Alarcon B. EMBO J. 1995; 14: 2257-2268Crossref PubMed Scopus (61) Google Scholar, 20Yang M. Ellenberg J. Bonifacino J.S. Weissman A.M. J. Biol. Chem. 1997; 272: 1970-1975Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 21Fu J. Kreibich G. J. Biol. Chem. 2000; 275: 3984-3990Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 22Parker A.K.T. Gergely F.V. Taylor C.W. J. Biol. Chem. 2004; 279: 23797-23805Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 23Wrzeszczynski K.O. Rost B. Cell. Mol. Life Sci. 2004; 61: 1341-1353Crossref PubMed Scopus (18) Google Scholar). The mechanism by which these motifs promote retention is unknown. Many ER-translocated proteins are covalently modified with a glycosylphosphatidylinositol (GPI) anchor (24Eisenhaber B. Bork P. Eisenhaber F. Protein Eng. 2001; 14: 17-25Crossref PubMed Scopus (147) Google Scholar). GPI anchor attachment is catalyzed by GPI transamidase (GPIT), a membrane-bound protein complex composed of at least five subunits: Gpi8, Gaa1, PIG-S, PIG-T, and PIG-U (25Hamburger D. Egerton M. Riezman H. J. Cell Biol. 1995; 129: 629-639Crossref PubMed Scopus (149) Google Scholar, 26Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar, 27Ohishi K. Inoue N. Kinoshita T. EMBO J. 2001; 20: 4088-4098Crossref PubMed Scopus (140) Google Scholar, 28Hong Y. Ohishi K. Kang J.Y. Tanaka S. Inoue N. Nishimura J. Maeda Y. Kinoshita T. Mol. Biol. Cell. 2003; 14: 1780-1789Crossref PubMed Scopus (95) Google Scholar, 29Fraering P. Imhof I. Meyer U. Strub J.M. van Dorsselaer A. Vionnet C. Conzelmann A. Mol. Biol. Cell. 2001; 12: 3295-3306Crossref PubMed Scopus (94) Google Scholar, 30Grimme S.J. Gao X.D. Martin P.S. Tu K. Tcheperegine S.E. Corrado K. Farewell A.E. Orlean P. Bi E. Mol. Biol. Cell. 2004; 15: 2758-2770Crossref PubMed Google Scholar). GPIT removes a C-terminal GPI-directing signal peptide from the translocated protein and attaches a preassembled GPI anchor via an amide linkage to the newly exposed C-terminal amino acid residue of the protein (31Udenfriend S. Kodukula K. Annu. Rev. Biochem. 1995; 64: 563-5917Crossref PubMed Scopus (436) Google Scholar, 32Sharma D. Vidugiriene J. Bangs J.D. Menon A.K. J. Biol. Chem. 1999; 274: 16479-16486Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Gpi8 is presumed to form the catalytic center of GPIT (33Meyer U. Benghezal M. Imhof I. Conzelmann A. Biochemistry. 2000; 39: 3461-3471Crossref PubMed Scopus (71) Google Scholar, 34Ohishi K. Nagamune K. Maeda Y. Kinoshita T. J. Biol. Chem. 2003; 278: 13959-13967Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), but all subunits are essential for function. Recent data suggest that Gaa1 and possibly PIG-U may play a role in recruiting GPI to GPIT (28Hong Y. Ohishi K. Kang J.Y. Tanaka S. Inoue N. Nishimura J. Maeda Y. Kinoshita T. Mol. Biol. Cell. 2003; 14: 1780-1789Crossref PubMed Scopus (95) Google Scholar, 35Vainauskas S. Menon A.K. J. Biol. Chem. 2004; 279: 6540-6545Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and it has been proposed that a β-propeller structure in the luminal domain of PIG-T gates access to the active site of Gpi8 (36Eisenhaber B. Maurer-Stroh S. Novatchkova M. Schneider G. Eisenhaber F. BioEssays. 2003; 25: 367-385Crossref PubMed Scopus (150) Google Scholar). The role of PIG-S is unknown. Gpi8 and PIG-T are type I transmembrane proteins; the other three subunits have multiple transmembrane spans. Recent studies show that human Gaa1 has a cytoplasmically oriented N terminus and spans the ER membrane seven times (37Vainauskas S. Maeda Y. Kurniawan H. Kinoshita T. Menon A.K. J. Biol. Chem. 2002; 277: 30535-30542Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar); the membrane topology of PIG-S and PIG-U remains to be experimentally determined. We are interested in understanding how the GPIT complex is assembled into a functional unit that is localized to the ER membrane. The individual subunits are presumably independently synthesized and integrated into the ER membrane and must remain in the ER long enough to encounter the other subunits in order to form the GPIT complex. The complex itself, after assembly, must remain in the ER in order to execute its catalytic function. Thus, individual subunits as well as the GPIT complex as a whole must be able to avoid entering the secretory pathway and exiting the ER. To identify the mechanism(s) by which the subunits of GPIT are localized to the ER, we chose initially to analyze Gaa1 and PIG-T. We used fluorescence microscopy and glycosidase digestion to analyze the subcellular localization of epitope-tagged variants of these two proteins as well as that of fusion proteins prepared using elements of Gaa1 or PIG-T combined with sequences from the Golgi protein α2,6-sialyltransferase or the plasma membrane-localized proteins transferrin receptor and Tac antigen. This “cut-and-paste” approach revealed that Gaa1 does not contain any dominant ER localization information, whereas PIG-T is localized to the ER by virtue of a signal contained within its TM domain. These data are discussed in the context of other recent examples of signal-mediated and signalless retention of ER-resident membrane proteins. Antibodies—Anti-Gpi8 and anti-Gaa1 rabbit polyclonal antibodies were generated as described previously (37Vainauskas S. Maeda Y. Kurniawan H. Kinoshita T. Menon A.K. J. Biol. Chem. 2002; 277: 30535-30542Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Affinity-purified rabbit anti-PIG-T antibody was a gift from Dr. Taroh Kinoshita (Osaka University). Mouse monoclonal anti-FLAG antibody M2 was purchased from Sigma; mouse monoclonal anti-V5 antibody was purchased from Invitrogen. Rabbit polyclonal anti-V5 antibody was a gift from Dr. Karen Colley (University of Illinois at Chicago). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs were from Promega (Madison, WI). Construction of Mammalian Expression Vectors—C-terminally V5-tagged Gaa1 (pEF/D1V5) and Gaa19-ST7–402 (previously called N19-ST) constructs were generated as described previously (37Vainauskas S. Maeda Y. Kurniawan H. Kinoshita T. Menon A.K. J. Biol. Chem. 2002; 277: 30535-30542Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To obtain the Gaa1(R→A) construct, sense RA (5′-cgggatccgccatgggcctcctgtcggacccggttgctgcggccgcgctcgcccgcctag-3′) and antisense GAV5 (5′-ctggctctagatgatggtggtggtggtg-3′) primers and pEF/D1V5 as template DNA were used for the PCR amplification. The amplified DNA product was digested with BamHI/XbaI and ligated into BamHI/XbaI pEF6/V-5 His vector (Invitrogen). C-terminally V5-tagged Gaa1282 and Gaa1282(R→ A) constructs were generated using the sense strand T7 primer and the antisense primer LPV5 (5′-ctggctctagatccaatgatgtccagtcc-3′) from the plasmids pEF/D1V5 and pEF/Gaa1(R→ A), respectively. PCR-amplified products were digested with BamHI/XbaI and ligated into BamHI/XbaI pEF6/V-5 His vector. To replace the N-terminal cytoplasmic part and TM domain of rat α2,6-sialyltransferase (ST; the construct we used encoded ST with tyrosine, rather than cysteine, at amino acid 123) with the amino-terminal cytoplasmic part and TM of Gaa1, two PCR fragments were generated: (i) from pEF/D1V5 vector using T7 and GN3 (5′-tgggaattcgaggcgcagcactagg-3′) primers and (ii) from pcDNA3.1/STTYR vector (a gift from Dr. Karen Colley, University of Illinois at Chicago) using primers STLUM (5′-gggcagaattcgagcgactatgaggccct-3′) and BGH (5′-caactagaaggcacagtcgagg-3′). The obtained PCR products were digested with BamHI/EcoRI and EcoRI/XbaI, respectively, and ligated into BamHI/XbaI pEF6/V-5 His vector. The resulting Gaa155-ST30–402 construct includes N-terminal sequence of the Gaa1 (aa 1–55), followed by Glu and Phe and then residues 30–402 of human ST, fused with V5 epitope and His6 sequence. The same strategy was used to generate other chimeric proteins. The Gaa1282-ST30–402 construct contains N-terminal sequence of the Gaa1 (aa 1–282) fused to ectodomain of the ST (aa 30–402). To obtain human transferrin receptor fused at its C terminus with V5 and His6 epitope tags, sense T7 promoter and antisense TFR3 (5′-gccctctagaaactcattgtcaatgtccca-3′) primers were used for PCR amplification from plasmid pSVT7, (bearing human transferrin receptor cDNA) as template DNA (a gift from Dr. Paul Gleeson, University of Melbourne, Australia). The resulting PCR product was digested with EcoRI/XbaI and ligated into EcoRI/XbaI pEF6/V-5 His vector. To replace the N-terminal part of human transferrin receptor (TfR) with the N-terminal cytoplasmic part of Gaa1, two PCR fragments were generated: (i) from pEF/D1V5 vector using T7 and GN3 (5′-tgggaattcgaggcgcagcactagg-3′) primers and (ii) from pSVT7/TfR vector using primers TFR62 (5′-tgggaattctgtagtggaagtatctgctatgg-3′) and TFR3. Amplified DNA products were digested with BamHI/EcoRI and EcoRI/XbaI, respectively, and ligated into BamHI/XbaI pEF6/V-5 His vector. The resulting Gaa119-TfR63–760 construct includes the N-terminal 19 residues of hGaa1, followed by Glu and Phe followed by residues 63–760 of the human transferrin receptor, fused with V5 epitope and His6 sequence. The same strategy was used to generate other chimeric proteins. The Gaa155-TfR87–760 construct contains N-terminal sequence of the Gaa1 (aa 1–55) fused to the ectodomain of TfR (aa 87–760). Human PIG-T cDNA was obtained from HeLa First Strand cDNA (Stratagene, La Jolla, CA) by PCR amplification with a sense primer PT5 (5′-ggaattcgccatggcggcggctatgccgctt-3′) and an antisense primer PT3 (5′-acagagtggggggacacctcg-3′). The PCR-amplified product was ligated into the mammalian expression vector pEF6/V5-His-TOPO (Invitrogen). The resulting pEF/PIG-T-V5 construct encodes PIG-T with C-terminal V5 and His6 epitope tags. To generate truncated PIG-T constructs T545-V5 and T514-FLAG, the primers T545 (5′-ggccctctagatcgatattgtagaaggagccatagca-3′) and T514 (5′-gtaatcgatgacctccgtgtagagccgcaca-3′) were used, respectively. To generate Tace-T516–578 construct, the ectodomain of Tac was obtained by PCR from pEF6/Tac plasmid (38Pottekat A. Menon A.K. J. Biol. Chem. 2004; 279: 15743-15751Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) using primers T7 and TACLUM3 (5′-gtaatcgattgtaaatatggacgtctccatg-3′), and the C-terminal part of PIG-T was amplified with primers T514F (5′-gtcatcgattacaacctgccgacaccggact-3′) and BGH. The obtained PCR products were digested with BamHI/ClaI and ClaI/XbaI, respectively, and ligated into BamHI/XbaI pEF6/V-5 His vector. Cell Culture and Transfection—HeLa cells were cultured at 37 °C in a humidified 5% CO2 atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum. Cells were transfected with cDNA constructs by electroporation as described previously (37Vainauskas S. Maeda Y. Kurniawan H. Kinoshita T. Menon A.K. J. Biol. Chem. 2002; 277: 30535-30542Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Immunoprecipitations and Immunoblots—Transfected HeLa cells (1–2 × 107 cells) were harvested by scraping 48 h post-transfection, washed once with PBS, resuspended in 1 ml of MSB buffer (20 mm Hepes-KOH, pH 7.6, 200 mm NaCl, 1% digitonin or Nonidet P-40, and 1× protease inhibitor mixture (Calbiochem)), and solubilized on ice for 30 min. The resulting cell lysate was clarified by centrifugation at 10,000 × g for 20 min at 4 °C. The 10,000 × g supernatant was incubated with 30 μl of anti-FLAG M2, or anti-V5 agarose (Sigma) slurry was added and incubated at 4 °C for 4 h with gentle agitation. The beads were pelleted by 15-s centrifugation at 10,000 × g. The samples were washed four times for 5 min each in 1.5 ml of buffer MSB. Bound antigen was released from the beads by incubation with FLAG or V5 peptide (200 μg/ml) in buffer MSB. Immunoprecipitated fractions were analyzed by SDS-PAGE, followed by immunoblotting using chemiluminescence reagents (Pierce). Pulse-Chase Labeling—Transfected HeLa cells were analyzed 48 h post-transfection. The cells were pulse-labeled for 10 min with EXPRE35S35S Protein Labeling Mix (1 mCi/ml; PerkinElmer Life Sciences) in methionine/cysteine-free complete medium and then chased for 2–8 h at 37 °C. Cell lysates were prepared, and immunoprecipitations were carried out as described above. Immunoprecipitated fractions were resolved by SDS-PAGE. After electrophoresis, the gels were dried and radiolabeled proteins were visualized and quantitated using a PhosphorImager (Amersham Biosciences) and ImageQuant software (Amersham Biosciences). Fluorescence Microscopy—Transfected HeLa cells were plated on glass coverslips, cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, fixed with 4% paraformaldehyde, permeabilized with 0.3% (w/v) Triton X-100, and incubated with primary and secondary antibodies as described previously (37Vainauskas S. Maeda Y. Kurniawan H. Kinoshita T. Menon A.K. J. Biol. Chem. 2002; 277: 30535-30542Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The labeled cells were visualized using a Bio-Rad confocal microscope (type MRC 1000). Endoglycosidase H (Endo H) and Peptide:N-Glycosidase F (PNGase F) Treatment—Endo H and PNGase F from New England Biolabs was used for carbohydrate digestion. The treatment was done as described previously (35Vainauskas S. Menon A.K. J. Biol. Chem. 2004; 279: 6540-6545Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). A Triple Arginine Motif in the Cytoplasmically Oriented N-terminal Sequence of Gaa1 Does Not Control ER Localization of the Protein—We previously suggested that the cytoplasmic N terminus of Gaa1 might function as an ER sorting determinant (37Vainauskas S. Maeda Y. Kurniawan H. Kinoshita T. Menon A.K. J. Biol. Chem. 2002; 277: 30535-30542Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Inspection of the primary sequence of human Gaa1 identified an internal triple arginine cluster (Arg9-Arg10-Arg11) near the N terminus (Fig. 1A). This arginine cluster resembles the Arg-Xaa-Arg ER localization motifs recently identified in subunits of multimeric protein complexes displayed at the cell surface (18Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar, 39Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 40Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (103) Google Scholar). Correct assembly of these protein complexes and their transport to the cell surface appears to be regulated through control of the trafficking of individual subunits, such that incompletely assembled complexes are retained in the ER. The secretory transport machinery is able to differentiate between unassembled units and assembled complexes because the Arg-Xaa-Arg motif in individual subunits can be masked after assembly of the protein into the complex or because the ability of the motif to signal ER retention/retrieval can be suppressed in some cases by phosphorylation of adjacent residues. We hypothesized that the Arg9-Arg10-Arg11 motif in Gaa1 acts as an ER localization signal. We tested this hypothesis by preparing a Gaa1 variant (denoted Gaa1(R→ A)) in which the three arginines were replaced with alanine residues. HeLa cells were transfected with plasmids encoding wild type, V5-epitope-tagged Gaa1 (Fig. 1A), or similarly tagged Gaa1(R→ A). The subcellular distribution of the transiently expressed proteins was determined by indirect immunofluorescence microscopy using anti-V5 antibodies. Fig. 1B shows that both wild type Gaa1 and Gaa1(R→ A) display a reticular staining pattern consistent with ER localization. Gaa1 and Gaa1(R→ A) were extracted from cells in detergent-containing buffer, treated with glycosidases, and analyzed by SDS-PAGE and immunoblotting. Fig. 1C shows that both proteins are modified with a single, endoglycosidase H-susceptible N-glycan, suggesting that the wild-type Gaa1 and Gaa1(R→ A) are not exposed to Golgi N-glycan modification enzymes. We considered the possibility that, without its arginine cluster, Gaa1(R→ A) could potentially be exported from the ER but is unable to leave the compartment as a result of improper folding. Misfolding would mitigate the ability of the protein to pass ER quality control systems for secretion and potentially accelerate its rate of turnover. Thus, compared with wild-type Gaa1, Gaa1(R→ A) may be turned over more rapidly by ER-associated degradation pathways. To test whether this was the case, we used [35S]methionine/cysteine to pulse-label HeLa cells that were transiently expressing wild-type Gaa1 or Gaa1(R→ A) and then chased the cells for various periods of time (up to 8 h), extracted proteins in detergent-containing buffer, and used immunoprecipitation, SDS-PAGE, and PhosphorImager analysis to determine the amount of radiolabeled Gaa1 or Gaa1(R→ A) remaining in the cells. The SDS-PAGE/PhosphorImager data for one experiment are shown in Fig. 1D, whereas the quantitation of the rate of decay taken from three independent experiments is shown in Fig. 1E. The data show that Gaa1(R→ A) turns over with a half-time of ∼3.2 h, similar to the rate of turnover of wild-type Gaa1 (a single, monoexponential decay curve is plotted for both proteins in Fig. 1E). This result suggests that it is unlikely that folding/degradation problems underlie the ER retention of Gaa1(R→ A). Together with the immunofluorescence localization of Gaa1(R→ A) to the ER and the Endo H-susceptibility of its N-glycan, these data suggest that the triple arginine motif (Arg9-Arg10-Arg11) is not necessary for ER localization of Gaa1. Gaa1282, a Construct Consisting of the N-terminal Cytoplasmic Tail, First TM Domain, and Part of the N-Glycosylated Luminal Loop of Gaa1, Is ER-localized—Co-immunoprecipitation experiments indicated that Gaa1(R→ A), like Gaa1, interacts with Gpi8, PIG-T, and PIG-S (data not shown). It is therefore conceivable that Gaa1(R→ A) is retained in the ER through its interactions with other subunits of the GPI transamidase and that these interactions render potential localization information in the triple arginine motif redundant. Alternatively, it is possible that full-length Gaa1 contains more than one ER localization signal, rendering individual signals redundant. To clarify this issue, we investigated the subcellular localization of Gaa1282, a truncated molecule consisting of the first 282 amino acids of Gaa1 capped with a C-terminal V5 epitope tag (Fig. 2A). Our choice of truncation site was based on previous results that indicated that a similar construct containing a larger stretch of the luminal loop (up to amino acid 367, right before the start of the second TM domain) was proteolytically cleaved in situ to yield a membrane-anchored fragment analogous to Gaa1282; this fragment was ER-localized but not able to bind to Gpi8, PIG-T, and PIG-S (37Vainauskas S. Maeda Y. Kurniawan H. Kinoshita T. Menon A.K. J. Biol. Chem. 2002; 277: 30535-30542Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In addition to Gaa1282,we generated a construct, Gaa1282(R→ A), in which the triple arginine cluster (Arg9-Arg10-Arg11) was altered to Ala9-Ala10-Ala11. Neither Gaa1282 nor Gaa1282(R→ A) is expected to interact with other transamidase subunits. We confirmed this via co-immunoprecipitation experiments that showed that the Gaa1282 constructs, unlike full-length Gaa1, could not co-immunoprecipitate endogenous Gpi8 or PIG-T (Fig. 2B). Co-immunoprecipitation experiments done on lysates prepared from cells that co-expressed the Gaa1282 constructs with green fluorescent protein-tagged PIG-T similarly demonstrated that the truncated proteins could not co-immunoprecipitate PIG-T-green fluorescent protein (data not shown). Indirect immunofluorescence microscopy showed that both Gaa1282 and Gaa1282(R→ A) were ER-localized (Fig. 2C), and glycosidase digestion showed that both possessed an Endo H-susceptible N-glycan (Fig. 2D). These results reflect the data obtained with full-length Gaa1 constructs (Fig. 1) and confirm that the arginine cluster in the N-terminal cytoplasmic tail of Gaa1 is not required for correct topological insertion of the protein or for its ER localization. Gaa1282 Does Not Contain a Dominant ER Retention Signal—Our results thus far eliminate a role for the triple arginine cluster (Arg9-Arg10-Arg11) in ER localization of Gaa1 and Gaa1282 b" @default.
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• W2166656822 title "Endoplasmic Reticulum Localization of Gaa1 and PIG-T, Subunits of the Glycosylphosphatidylinositol Transamidase Complex" @default.
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• W2166656822 doi "https://doi.org/10.1074/jbc.m414253200" @default.
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