Structural Requirements for the Recruitment of Gaa1 into a Functional Glycosylphosphatidylinositol Transamidase Complex
2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês
10.1074/jbc.m205402200
ISSN1083-351X
AutoresSaulius Vainauskas, Yusuke Maeda, Henry Kurniawan, Taroh Kinoshita, Anant K. Menon,
Tópico(s)Cellular transport and secretion
ResumoGlycosylphosphatidylinositol (GPI)-anchored proteins are synthesized on membrane-bound ribosomes, translocated across the endoplasmic reticulum membrane, and GPI-anchored by GPI transamidase (GPIT). GPIT is a minimally heterotetrameric membrane protein complex composed of Gaa1, Gpi8, PIG-S and PIG-T. We describe structure-function analyses of Gaa1, the most hydrophobic of the GPIT subunits, with the aim of assigning a functional role to the different sequence domains of the protein. We generated epitope-tagged Gaa1 mutants and analyzed their membrane topology, subcellular distribution, complex-forming capability, and ability to restore GPIT activity in Gaa1-deficient cells. We show that (i) detergent-extracted, Gaa1-containing GPIT complexes sediment unexpectedly rapidly at ∼17 S, (ii) Gaa1 is an endoplasmic reticulum-localized membrane glycoprotein with a cytoplasmically oriented N terminus and a lumenally oriented C terminus, (iii) elimination of C-terminal transmembrane segments allows Gaa1 to interact with other GPIT subunits but renders the resulting GPIT complex nonfunctional, (iv) interaction between Gaa1 and other GPIT subunits occurs via the large lumenal domain of Gaa1 located between the first and second transmembrane segments, and (v) the cytoplasmic N terminus of Gaa1 is not required for formation of a functional GPIT complex but may act as a membrane-sorting determinant directing Gaa1 and associated GPIT subunits to an endoplasmic reticulum membrane domain. Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized on membrane-bound ribosomes, translocated across the endoplasmic reticulum membrane, and GPI-anchored by GPI transamidase (GPIT). GPIT is a minimally heterotetrameric membrane protein complex composed of Gaa1, Gpi8, PIG-S and PIG-T. We describe structure-function analyses of Gaa1, the most hydrophobic of the GPIT subunits, with the aim of assigning a functional role to the different sequence domains of the protein. We generated epitope-tagged Gaa1 mutants and analyzed their membrane topology, subcellular distribution, complex-forming capability, and ability to restore GPIT activity in Gaa1-deficient cells. We show that (i) detergent-extracted, Gaa1-containing GPIT complexes sediment unexpectedly rapidly at ∼17 S, (ii) Gaa1 is an endoplasmic reticulum-localized membrane glycoprotein with a cytoplasmically oriented N terminus and a lumenally oriented C terminus, (iii) elimination of C-terminal transmembrane segments allows Gaa1 to interact with other GPIT subunits but renders the resulting GPIT complex nonfunctional, (iv) interaction between Gaa1 and other GPIT subunits occurs via the large lumenal domain of Gaa1 located between the first and second transmembrane segments, and (v) the cytoplasmic N terminus of Gaa1 is not required for formation of a functional GPIT complex but may act as a membrane-sorting determinant directing Gaa1 and associated GPIT subunits to an endoplasmic reticulum membrane domain. glycosylphosphatidylinositol GPI transamidase endoplasmic reticulum phosphatidylinositol glycan α2,6-sialyltransferase transmembrane phosphate-buffered saline endoglycosidase H Chinese hamster ovary Glycosylphosphatidylinositol (GPI)1-anchored proteins are synthesized as prepro-proteins with an endoplasmic reticulum (ER)-targeting N-terminal signal sequence and a C-terminal signal sequence that directs the attachment of the GPI anchor (1Udenfriend S. Kodukula K. Annu. Rev. Biochem. 1995; 64: 563-591Crossref PubMed Scopus (437) Google Scholar, 2Kodukula K. Maxwell S.E. Udenfriend S. Methods Enzymol. 1995; 250: 536-547Crossref PubMed Scopus (17) Google Scholar, 3Eisenhaber B. Bork P. Eisenhaber F. Protein Eng. 2001; 14: 17-25Crossref PubMed Scopus (151) Google Scholar, 4Kinoshita T. Inoue N. Curr. Opin. Chem. Biol. 2000; 4: 632-638Crossref PubMed Scopus (171) Google Scholar, 5McConville M.J. Menon A.K. Mol. Membr. Biol. 2000; 17: 1-16Crossref PubMed Scopus (127) Google Scholar). Upon translocation of the prepro-protein across the ER membrane, the N-terminal signal sequence is removed by signal peptidase resulting in a pro-protein. The pro-protein is then processed by GPI transamidase (GPIT), a novel multisubunit enzyme that removes the C-terminal signal sequence and attaches a GPI molecule to the newly exposed C-terminal amino acid (6Mayor S. Menon A.K. Cross G.A.M. J. Cell Biol. 1991; 114: 61-71Crossref PubMed Scopus (63) Google Scholar, 7Maxwell S.E. Ramalingam S. Gerber L.D. Brink L. Udenfriend S. J. Biol. Chem. 1995; 270: 19576-19582Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Sharma 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). The linkage between GPI and protein is an amide bond between the capping ethanolamine residue in the GPI structure and the C-terminal α-carboxylic acid of the protein (9Holder A.A. Biochem. J. 1983; 209: 261-262Crossref PubMed Scopus (61) Google Scholar). The GPIT-catalyzed reaction represents the final step in the assembly of a GPI-anchored protein and provides the critical post-translational modification of this class of proteins that allows them to gain entry into ER-derived transport vesicles for delivery to the cell surface (10Doering T.L. Schekman R. EMBO J. 1996; 15: 182-191Crossref PubMed Scopus (115) Google Scholar, 11Field M.C. Moran P., Li, W. Keller G.A. Caras I.W. J. Biol. Chem. 1994; 269: 10830-10837Abstract Full Text PDF PubMed Google Scholar). GPIT is a minimally heterotetrameric, membrane-bound protein complex containing the subunits Gpi8 (∼45 kDa), Gaa1 (∼67 kDa), PIG-S (∼62 kDa), and PIG-T (∼65 kDa). Gaa1 and Gpi8 were identified through genetic studies in yeast (12Hamburger D. Egerton M. Riezman H. J. Cell Biol. 1995; 129: 629-639Crossref PubMed Scopus (149) Google Scholar, 13Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelman A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar), whereas PIG-S and PIG-T were identified more recently through co-immunoprecipitation experiments using epitope-tagged Gpi8 (14Ohishi K. Inoue N. Kinoshita T. EMBO J. 2001; 20: 4088-4098Crossref PubMed Scopus (143) Google Scholar, 15Fraering P. Imhof I. Meyer U. Strub J.M. van Dorsselaer A. Vionnet C. Conzelmann A. Mol. Biol. Cell. 2001; 12: 3295-3306Crossref PubMed Scopus (95) Google Scholar). Photocross-linking experiments indicate that the complex may contain at least one other protein with a molecular mass of ∼120 kDa that appears not to associate with the other components under immunoprecipitation conditions (16Vidugiriene J. Vainauskas S. Johnson A.E. Menon A.K. Eur. J. Biochem. 2001; 268: 2290-2300Crossref PubMed Scopus (32) Google Scholar). Gpi8, Gaa1, PIG-S, and PIG-T are all required for transamidase function (12Hamburger D. Egerton M. Riezman H. J. Cell Biol. 1995; 129: 629-639Crossref PubMed Scopus (149) Google Scholar, 13Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelman A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar, 14Ohishi K. Inoue N. Kinoshita T. EMBO J. 2001; 20: 4088-4098Crossref PubMed Scopus (143) Google Scholar, 15Fraering P. Imhof I. Meyer U. Strub J.M. van Dorsselaer A. Vionnet C. Conzelmann A. Mol. Biol. Cell. 2001; 12: 3295-3306Crossref PubMed Scopus (95) Google Scholar,17Ohishi K. Inoue N. Maeda Y. Takeda J. Riezman H. Kinoshita T. Mol. Biol. Cell. 2000; 11: 1523-1533Crossref PubMed Scopus (108) Google Scholar, 18Yu J. Nagarajan S. Knez J.J. Udenfriend S. Chen R. Medof M.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12580-12585Crossref PubMed Scopus (66) Google Scholar). Gpi8 appears to be the likely enzymatic component of the GPIT protein complex because it shares sequence homology with a family of plant vacuolar endopeptidases, one of which catalyzes the transamidation step in the maturation of concanavalin A (13Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelman A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar, 19Meyer U. Benghezal M. Imhof I. Conzelmann A. Biochemistry. 2000; 39: 3461-3471Crossref PubMed Scopus (72) Google Scholar). Gaa1, PIG-S, and PIG-T share no homology with any proteins of known function, and their functional role in GPIT action is unclear. There is no “soluble” assay for GPIT. Most measures of GPIT activity described thus far require the ER protein translocation apparatus and an intact ER membrane to generate an appropriate pro-protein substrate (2Kodukula K. Maxwell S.E. Udenfriend S. Methods Enzymol. 1995; 250: 536-547Crossref PubMed Scopus (17) Google Scholar, 20Kodukula K. Micanovic R. Gerber L. Tamburrini M. Brink L. Udenfriend S. J. Biol. Chem. 1991; 266: 4464-4470Abstract Full Text PDF PubMed Google Scholar, 21Vidugiriene J. Menon A.K. EMBO J. 1995; 14: 4686-4694Crossref PubMed Scopus (25) Google Scholar, 22Doering T.L. Schekman R. Biochem. J. 1997; 328: 669-675Crossref PubMed Scopus (17) Google Scholar). Isolated Gpi8 shows no activity against a variety of protein and peptide substrates (13Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelman A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar). Nevertheless, photocross-linking experiments show that Gpi8, Gaa1, a ∼120-kDa protein, and possibly PIG-S are in close physical proximity to the pro-protein substrates of GPIT as they undergo processing (16Vidugiriene J. Vainauskas S. Johnson A.E. Menon A.K. Eur. J. Biochem. 2001; 268: 2290-2300Crossref PubMed Scopus (32) Google Scholar, 23Spurway T.D. Dalley J.A. High S. Bulleid N.J. J. Biol. Chem. 2001; 276: 15975-15982Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Other analyses using a mammalian cell-free translation-translocation system indicate that pro-proteins bind Gaa1 in the absence of Gpi8 but that binding of pro-proteins to Gpi8 does not occur in the absence of Gaa1. 2R. Chen and M. E. Medof, submitted for publication. Although these studies are suggestive, it is difficult to take them further to analyze the physical and functional architecture of GPIT and determine the role of its various constituent subunits. In an attempt to dissect the GPIT complex from a structure-function perspective, we opted to analyze human Gaa1. Gaa1 is the most conspicuously hydrophobic of the known subunits of GPIT, and the results described above suggest that it may play a key role in substrate recognition. It is predicted to be a multispanning membrane protein of ∼67 kDa with seven transmembrane (TM) domains, a large lumenal domain between the first two TM segments, and a cytoplasmically oriented N terminus bearing a potential ER retrieval signal in the form of a di-arginine motif (24Teasdale R.D. Jackson M.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 27-54Crossref PubMed Scopus (448) Google Scholar). The TM domains may play a role in recognizing the hydrophobic component of the GPI signal sequence in pro-proteins, whereas the lumenal domain may mediate the association of Gaa1 with the other GPIT subunits and possibly also GPI. Furthermore, the potential ER retrieval signal in Gaa1 may serve to localize the entire human GPIT complex to the ER because no known ER retention/retrieval motifs are evident in any of the other subunits of mammalian GPIT. To explore these possibilities, we created a series of epitope tagged C-terminal deletion variants of human Gaa1, expressed them in mammalian cells, and used them to analyze the role of Gaa1 in GPIT. Our results provide a clear functional delineation of the different sequence domains of Gaa1. Dulbecco's modified Eagle's medium, fetal bovine serum, and penicillin-streptomycin were purchased from Invitrogen (San Diego, CA). Goat serum was purchased from Sigma. Restriction enzymes, DNA modifying enzymes, and DNA polymerase were purchased from MBI Fermentas and New England Biolabs. All of the other chemicals were of reagent grade and were used without further purification. Anti-Gpi8 and anti-Gaa1 rabbit polyclonal antibodies were generated against Escherichia coli-expressed polypeptides corresponding to residues 31–322 of human Gpi8 and residues 60–205 of human Gaa1, respectively. Mouse monoclonal antibodies against the FLAG epitope (an 8-amino acid sequence consisting of DYKDDDDK), the V5 epitope (a 14-amino acid sequence consisting of GKPIPNPLLGLDST), and human calnexin were purchased from Sigma, Invitrogen, and Transduction Laboratories (Lexington, KY), respectively. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs were from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse and anti-rabbit IgGs conjugated with Alexa Fluor 568 or Alexa Fluor 488 were from Molecular Probes (Eugene, OR). N-terminally FLAG-tagged human Gaa1 cDNA subcloned into the pME18Sf vector (17Ohishi K. Inoue N. Maeda Y. Takeda J. Riezman H. Kinoshita T. Mol. Biol. Cell. 2000; 11: 1523-1533Crossref PubMed Scopus (108) Google Scholar) was used as a template to generate Gaa1 truncation mutants by PCR (see Figs. 3A and 8Afor schematic figures of the mutants). For the production of constructs D1–D7, primers were designed so that the sense strand primer TR9 included an EcoRI site at the 5′ end for subcloning, and the antisense primers D1–D7 included nontemplated sequences encoding the His6 epitope tag, a stop codon, and an XbaI restriction site at the 3′ end. After EcoRI and XbaI digestion, the PCR products were ligated toEcoRI/XbaI-digested pME18Sf vector. The resulting plasmids were named pME/D1–7. The Gaa1 open reading frame encompasses codons 1–621 (D1), 564 (D2), 524 (D3), 483 (D4), 446 (D5), 386 (D6), and 367 (D7). To generate Gaa1 constructs bearing a V5 epitope tag C-terminal to the His6 sequence, sense GN5 and antisense GAV5 primers with BamHI and XbaI sites, respectively, were used for PCR amplification of Gaa1 cDNA from pME18Sf/Gaa1. The PCR product was digested withBamHI/XbaI and ligated into aBamHI/XbaI pEF6/V5 His vector (Invitrogen). The resulting plasmid was named pEF/D1V5. The D10, D11, and D12 constructs were made by PCR using the sense strand primer D10 containing a SalI site and antisense primers D6, D7, and D1, respectively. PCR-amplified products were digested withSalI/XbaI and ligated toSalI/XbaI-digested pME18Sf/Gaa1 vector. In the resulting constructs 18 residues at the N terminus of Gaa1 were replaced with Met-FLAG tag epitope, followed by a 3-amino acid (Val-Asp-Arg) linker to the first TM segment.FIG. 8Analyses of Gaa1 variants lacking the cytoplasmically oriented N-terminal region. A, schematic of the epitope-tagged, N-terminally deleted human Gaa1 truncation constructs used in this study. The full-length, nontruncated D1 construct is shown for comparison. Black boxes, membrane-spanning segments; hatched boxes, FLAG tag;open boxes, cytoplasmically oriented N-terminal region of Gaa1; open triangles, His6 tag. B, SDS-PAGE and immunoblotting analysis of Gaa1 constructs expressed in HeLa cells. D6 and D7 (see Fig. 3A) have an intact N terminus; D10 and D11 are N-terminally deleted versions of D6 and D7. Immunoblots were carried out with anti-FLAG antibodies (top panel), anti-Gpi8 antibodies (middle panel), and anti-α-tubulin antibodies (lower panel). C, GAA1-deficient CHO cells transfected with an empty vector or the Gaa1 construct D12 were analyzed 2 days after transfection for surface expression of GPI-anchored CD59 by flow cytometry. Expression of D12 was monitored by immunoprecipitation and immunoblotting with anti-FLAG antibodies as in Fig. 7. The band marked with anasterisk is nonspecific.View Large Image Figure ViewerDownload (PPT) All substitutions of amino acid codons to Ala within the Gaa1 coding region in constructs pME/D24 (amino acids in positions 241–244 substituted with Ala residues) and pME/D35 (positions 354–358 substituted with Ala residues) were introduced by the primer-mediated mutagenesis method (25Higuchi R. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. Recombinant PCR: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA1990: 177-183Google Scholar). In both cases, pME/D1 was used as the amplification template. PCR products with substitution mutations were digested by EcoRI/XbaI and subcloned intoEcoRI/XbaI-digested pME18Sf vector. To replace the N-terminal portion of α2,6-sialyltransferase (ST) with the N-terminal cytoplasmic portion of Gaa1, a PCR fragment was generated from pME18Sf/Gaa1 vector using primers GN5 and GN3. The PCR product thus obtained was digested withBamHI/EcoRI and ligated intoBamHI/EcoRI Iip33-ST-V5-pcDNA 3.1 (a gift from Dr. Karen Colley, University of Illinois). The resulting N19-ST construct includes 19 N-terminal residues of human Gaa1, followed by Glu and Phe, and then the ST transmembrane region, stem region, and catalytic domain, fused with V5 epitope and His6sequence. 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. The cells were passaged every 3–4 days. Exponentially growing cells were harvested by trypsinization and washed once with cytomix buffer (26van den Hoff M.J. Moorman A.F. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Crossref PubMed Scopus (388) Google Scholar). The cells were subsequently resuspended at a density of 1 × 107 cells/ml in the same buffer, and 400 μl of suspension were transferred to a 0.4-cm electroporation cuvette (Invitrogen) on ice. 50 μg of plasmid DNA was added to the suspension in the cuvette and mixed well. The mixture was then exposed to a single electric pulse of 300 V with a capacitance of 1,000 microfarads using an Invitrogen gene pulser system. The cuvette was immediately placed on ice for 10 min, and the cells were then suspended in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and plated onto 100-mm plates. The cells were incubated at 37 °C in a 5% CO2 atmosphere for 48 h prior to harvesting for biochemical analyses and/or indirect immunofluorescence microscopy. 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 0.5% Nonidet P-40, and 1× protease inhibitor mixture (Calbiochem, San Diego, CA)), and solubilized on ice for 30 min. The cell lysates were clarified by centrifugation (10,000 ×g for 20 min at 4 °C). S10 supernatants were centrifuged at 100,000 × g for 45 min at 4 °C. To each supernatant fraction, 30 μl of anti-FLAG M2 agarose (Sigma) slurry was added, and the sample was incubated at 4 °C for 4 h with gentle agitation. The agarose beads were pelleted by 15 s of centrifugation at 10,000 × g. The samples were washed four times for 5 min each time in 1.5 ml of buffer MSB with 10 mm dithiothreitol. Bound antigen was released from the anti-FLAG M2 agarose beads by incubation with FLAG peptide (200 μg/ml) in MSB buffer. Immunoprecipitated fractions were subjected to centrifugation on a sucrose gradient or directly analyzed by SDS-PAGE, followed by immunoblotting using chemiluminescence reagents (Pierce). Sucrose gradients (4 ml) were centrifuged at 4 °C for 18 h at 192,000 × g, using a Beckman SW50.1 rotor. Following centrifugation, the gradients were fractionated into 350-μl aliquots, and the proteins were detected by SDS-PAGE and immunoblotting. Gradient performance and resolution were evaluated by analyzing standard proteins: bovine serum albumin (65 kDa, 4.2 S), yeast alcohol dehydrogenase (150 kDa, 7.6 S), catalase (250 kDa, 11 S), and ferritin (460 kDa, 17.7 S). The standard proteins were detected by SDS-PAGE/Coomassie staining. Transiently transfected HeLa cells were plated onto poly-d-lysine-coated glass coverslips and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. After 24 h the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After three more washes with PBS, the plasma membrane was selectively permeabilized with digitonin at 3 μg/ml in cytomix buffer with 0.3 m sucrose for 5 min on ice. Alternatively, plasma membranes as well as intracellular membranes were permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. After permeabilization, the cells were washed three times with PBS and incubated with 10% goat serum albumin in PBS for 60 min at room temperature to block nonspecific binding. The cells were then incubated for 1 h at room temperature with anti-FLAG monoclonal antibody at 1 μg/ml, anti-V5 monoclonal antibody at 1:500 dilution, or anti-calnexin monoclonal antibody at 1:1000 dilution, after which they were washed three times with PBS. Alexa Fluor 568 or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500 dilution) was then added, and the cells were incubated for 1 h at room temperature. After four washes with PBS at room temperature, the coverslips were mounted onto glass slides (a drop of Vectashield (Vector Laboratories, Burlingame, CA) was included during mounting of the coverslip to prevent rapid photobleaching of the fluorescent conjugates) and taken for confocal microscopy using a Bio-Rad confocal microscope (type MRC 1000) with optical section chosen at 0.2 μm/section. In vitro translation reactions were performed using TNT Quick Coupled Transcription System (Promega, Madison, WI) as described by the manufacturer. Endoglycosidase H (Endo H) from New England Biolabs was used for carbohydrate digestion. The protein samples were denatured by the addition of 0.1 volume of 10× Endo H denaturation buffer (5% SDS, 10% β-mercaptoethanol), followed by incubation for 5 min at 100 °C. Then 0.1 volume of 10× Endo H reaction buffer (0.5 m sodium citrate, pH 5.5, at 25 °C) was added to the denatured sample, followed by incubation with 1 μl of 4,000 units/μl Endo H for 1 h at 37 °C. The samples were analyzed by SDS-PAGE to monitor molecular mass reductions accompanying deglycosylation. Mouse GAA1 knockout F9 cells (1 × 107) (17Ohishi K. Inoue N. Maeda Y. Takeda J. Riezman H. Kinoshita T. Mol. Biol. Cell. 2000; 11: 1523-1533Crossref PubMed Scopus (108) Google Scholar) suspended in 400 μl of culture medium (high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum) were electroporated with 25 μg each of plasmids at 500 microfarads and 250 V using a Gene Pulser (Bio-Rad). Two days after electroporation, the cells were stained with biotinylated anti-Thy-1 G7 antibody followed by phycoerythrin-conjugated streptavidin (Biomeda, Foster City, CA) and analyzed by FACScan (Becton Dickinson, San Jose, CA). GAA1-deficient CHO D9PA91 cells (1 × 107) (27Abrami L. Fivaz M. Kobayashi T. Kinoshita T. Parton R.G. van der Goot F.G. J. Biol. Chem. 2001; 276: 30729-30736Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) were electroporated with 25 μg each of plasmids at 960 microfarads and 280V. Two days after the transfection, the cells were harvested, one-thirtieth of the sample was used for flow cytometric analysis, and the rest of the sample was used for immunoblotting. For cytometric analysis the cells were stained with biotinylated anti-CD59 5H8 antibody followed by phycoerythrin-conjugated streptavidin (Biomeda) and analyzed using a FACScan (Becton Dickinson). For immunoblotting analysis the cells were solubilized in 1 ml of buffer containing 20 mm Tris, pH 7.4, 150 mm NaCl, 1 mmEDTA, 1% Triton X-100, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride for 30 min on ice. The lysates obtained by centrifugation of the cells at 18,000 ×g for 20 min were incubated with 10 μl of M2 anti-FLAG beads overnight. The precipitates were washed five times with the same buffer without protease inhibitors, heated with sample buffer for SDS-PAGE, and analyzed by immunoblotting using biotinylated M2 anti-FLAG antibody and horseradish peroxidase-conjugated streptavidin (Amersham Biosciences). The proteins were resolved by SDS-PAGE using polyacrylamide slab gels made according to the method of Laemmli (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212375) Google Scholar). Protein sequence analysis was performed at the Harvard Microchemistry Facility (golgi.harvard.edu/microchem/) by microcapillary reverse-phase high pressure liquid chromatography nanoelectrospray tandem mass spectrometry on a Finnegan LCQ DECA quadrupole ion trap mass spectrometer. The predicted molecular mass of a stoichiometric complex of the four known components of GPIT is ∼240 kDa. To assess the size of the native GPIT complex directly, a digitonin extract of HeLa cell microsomes was analyzed by velocity sedimentation on a 5–30% sucrose gradient. Immunoblotting of the gradient fractions with anti-Gpi8 and anti-Gaa1 antibodies (Fig. 1A) showed a fast-sedimenting pattern centered roughly around the ∼17.7 S (∼460 kDa) apo-ferritin standard. A similar analysis of a denaturing SDS extract showed Gpi8 sedimenting at ∼4 S, consistent with its ∼45-kDa monomeric size. These data indicate that GPIT sediments as a ∼17 S complex, larger than expected for a globular protein complex of ∼240 kDa. Our results are consistent with recent blue native gel electrophoresis analyses in which yeast Gpi8p was found within high molecular mass complexes in the 430–650-kDa range (15Fraering P. Imhof I. Meyer U. Strub J.M. van Dorsselaer A. Vionnet C. Conzelmann A. Mol. Biol. Cell. 2001; 12: 3295-3306Crossref PubMed Scopus (95) Google Scholar). The large sedimentation coefficient of the digitonin-extracted GPIT complex may be attributed to a nonglobular shape, bound detergent, the presence of multiple copies of one or more of the known subunits, the presence of additional, hitherto unidentified subunits, and/or interaction with other cellular components (29Tanford C. Nozaki Y. Reynolds J.A. Makino S. Biochemistry. 1974; 13: 2369-2376Crossref PubMed Scopus (381) Google Scholar). We investigated the latter possibilities by expressing FLAG-tagged human Gaa1 in HeLa cells and using anti-FLAG antibodies to immunoprecipitate FLAG-Gaa1 complexes from a digitonin or Nonidet P-40 extract of the cells. Cells transfected with an empty vector were used to assess the specificity of the immunoprecipitation. Fig. 1B shows that PIG-S, PIG-T, Gpi8, and α- and β-tubulin are all co-immunoprecipitated with FLAG-Gaa1. The presence of the tubulin isoforms as well as PIG-S and PIG-T was confirmed by nanoelectrospray tandem mass spectrometric analysis of tryptic peptides. The presence of tubulin in the immunoprecipitates is discussed below. Several other protein bands were also seen in the FLAG-Gaa1 immunoprecipitate, but these were deemed to be nonspecific because they were either found in control immunoprecipitates or seen to sediment separately from the immunoprecipitated GPIT complex on sucrose velocity gradients (see below). The lower intensity of bands corresponding to PIG-S, PIG-T, and Gpi8 compared with Gaa1 (Fig. 1B) is most likely due to the fact that FLAG-Gaa1 is overexpressed, and not all copies of the protein are integrated into GPIT complexes (see Fig. 4 and accompanying text). Other possible explanations are that the complex is not a stoichiometric combination of the four known subunits or that the subunits are not equally well stained with silver. As a prelude to our mutagenesis study of Gaa1, we carried out experiments to verify the ER localization and membrane topology of the protein. Based upon predictive algorithms, Gaa1 is presumed to be an integral membrane protein with 7 TM domains and a large loop between the first and second TM segments. The N terminus of the protein is predicted to be oriented toward the cytoplasm, whereas the C terminus is expected to be oriented toward the ER lumen. Gaa1 constructs containing either an N-terminal FLAG epitope tag or a C-terminal V5 epitope tag were expressed in HeLa cells. Both expressed constructs displayed a reticular distribution pattern characteristic of the ER. The cells were fixed, treated with digitonin to permeabilize the plasma membrane or Triton X-100 to permeabilize all membranes, and labeled with antibodies to the FLAG or V5 epitope tags. As shown in Fig. 2, digitonin-permeabilized cells could be labeled with anti-FLAG antibodies, confirming the cytoplasmic orientation of the Gaa1 N terminus. In contrast, the V5 epitope tag could be labeled only after Triton X-100 permeabilization, implying that the Gaa1 C terminus is sequestered in the lumen of the ER. These data, together with evidence that an asparagine residue within a glycosylation sequon in the loop region of Gaa1 is N-glycosylated (data not shown, but see corresponding data for truncated Gaa1 variants below), indicate that the predicted topology of the protein is correct. To identify structural features of Gaa1 required for its interaction with the other known components of GPIT, we created a series of N-terminally FLAG-tagged truncation mutants (Fig.3A) in which we systematically removed six of the seven predicted membrane-spanning segments from the C terminus of Gaa1. These constructs (termed D2–D7), as well as similarly tagged full-length Gaa1 (termed D1), were transiently expressed in HeLa cells. The cells were extracted with Nonidet P-40, and the detergent extracts were incubated with anti-FLAG-agarose beads to immunoprecipitate the Gaa1 constructs and any associated proteins. Immunoblotting with anti-FLAG antibodies indicated that all constructs were well expressed, with the possible exception of D2 and D5, which were expressed at lower levels compared with D1 (Fig. 3B, upper panel). Immunoblotting with anti-Gpi8 antibodies indicated that Gpi8 was immunoprecipitated with all of the Gaa1 constructs except D7, a construct representing only the first N-terminal TM domain and lumenal loop (Fig. 3B, lower panel). Silve
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