Biogenesis and Topology of the Transient Receptor Potential Ca2+ Channel TRPC1
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m312456200
ISSN1083-351X
AutoresYoko Dohke, Young S. Oh, Indu S. Ambudkar, R Turner,
Tópico(s)Cellular transport and secretion
ResumoThe TRPC ion channels are candidates for the store-operated Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. Hydropathy analyses indicate that these proteins contain eight hydrophobic regions (HRs) that could potentially form α-helical membrane-spanning segments. Based on limited sequence similarities to other ion channels, it has been proposed that only six of the eight HRs actually span the membrane and that the last two membrane-spanning segments (HRs 6 and 8) border the ion-conducting pore of which HR 7 forms a part. Here we study the biogenesis and transmembrane topology of human TRPC1 to test this model. We have employed a truncation mutant approach combined with insertions of glycosylation sites into full-length TRPC1. In our truncation mutants, portions of the TRPC1 sequence containing one or more HRs were fused between the enhanced green fluorescent protein and a C-terminal glycosylation tag. These chimeras were transiently expressed in the human embryonic cell line HEK-293T. Glycosylation of the tag was used to monitor its location relative to the lumen of the endoplasmic reticulum and thereby HR orientation. Our data indicate that HRs 1, 4, and 6 cross the membrane from cytosol to the ER lumen, that HRs 2, 5, and 8 have the opposite orientation, and that HR 3 is left out of the membrane on the cytosolic side. Our results also show that the sequence downstream of HR 8 plays an important role in anchoring its C-terminal end on the cytosolic side of the membrane. This effect appears to prevent HR 7 from spanning the bilayer and to result in its forming a pore-like structure of the type previously envisioned for the TRPC channels. We speculate that a similar mechanism may be responsible for the formation of other ion channel pores. The TRPC ion channels are candidates for the store-operated Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. Hydropathy analyses indicate that these proteins contain eight hydrophobic regions (HRs) that could potentially form α-helical membrane-spanning segments. Based on limited sequence similarities to other ion channels, it has been proposed that only six of the eight HRs actually span the membrane and that the last two membrane-spanning segments (HRs 6 and 8) border the ion-conducting pore of which HR 7 forms a part. Here we study the biogenesis and transmembrane topology of human TRPC1 to test this model. We have employed a truncation mutant approach combined with insertions of glycosylation sites into full-length TRPC1. In our truncation mutants, portions of the TRPC1 sequence containing one or more HRs were fused between the enhanced green fluorescent protein and a C-terminal glycosylation tag. These chimeras were transiently expressed in the human embryonic cell line HEK-293T. Glycosylation of the tag was used to monitor its location relative to the lumen of the endoplasmic reticulum and thereby HR orientation. Our data indicate that HRs 1, 4, and 6 cross the membrane from cytosol to the ER lumen, that HRs 2, 5, and 8 have the opposite orientation, and that HR 3 is left out of the membrane on the cytosolic side. Our results also show that the sequence downstream of HR 8 plays an important role in anchoring its C-terminal end on the cytosolic side of the membrane. This effect appears to prevent HR 7 from spanning the bilayer and to result in its forming a pore-like structure of the type previously envisioned for the TRPC channels. We speculate that a similar mechanism may be responsible for the formation of other ion channel pores. The TRPC ion channels are a family of Ca2+-permeable cation channels that are activated following receptor-mediated stimulation of phospholipase C (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (967) Google Scholar, 2Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Crossref PubMed Scopus (534) Google Scholar, 3Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar). The TRPC family belongs to the TRP superfamily of non-voltage-gated cation channels that also includes channels involved in pain transduction, epithelial Ca2+ transport, osmoregulation, mechanosensitivity, cell growth and differentiation, and other as yet uncharacterized functions (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (967) Google Scholar, 2Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Crossref PubMed Scopus (534) Google Scholar, 3Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar). At least 20 TRP superfamily members including seven TRPC family members (TRPC1–7) have thus far been identified in mammals. Recent studies indicate that members of the TRPC family are candidates for the store-operated Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores, a pathway whose molecular identity has remained elusive. However, the details of their involvement in this process and the downstream mechanisms underlying their activation following store depletion remain controversial (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (967) Google Scholar, 2Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Crossref PubMed Scopus (534) Google Scholar, 3Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar). Our understanding of the function and regulation of this important class of ion channels as well as the design and interpretation of experiments to probe their structure/function relationships require information concerning their transmembrane topology. There is strong evidence that the N and C termini of the TRPC channels are intracellular (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (967) Google Scholar, 2Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Crossref PubMed Scopus (534) Google Scholar, 3Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar, 4Vannier B. Zhu X. Brown D. Birnbaumer L. J. Biol. Chem. 1998; 273: 8675-8679Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and hydropathy analyses indicate the presence of eight hydrophobic regions (HRs) 1The abbreviations used are: HR, hydrophobic region; ER, endoplasmic reticulum; MSS, membrane-spanning segment; EGFP, enhanced green fluorescent protein; PNGase F, peptide N-glycosidase F; HA, hemagglutinin; HEK, human embryonic kidney; lum, luminal; Cyt, cytosol. that could potentially form α-helical membrane-spanning segments (MSSs). Based on sequence similarities to other apparently structurally related ion channels (5Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (387) Google Scholar), it is thought that only six of the eight HRs actually form MSSs. Specifically, four of the first five HRs are thought to span the membrane, HRs 6 and 8 are thought to span the membrane and border the ion-conducting pore, and HR 7 is thought to form a part of the pore by dipping into the membrane from the extracellular surface (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (967) Google Scholar, 2Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Crossref PubMed Scopus (534) Google Scholar, 3Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar). Evidence supporting this general topology scheme has been obtained for human TRPC3 by Vannier et al. (4Vannier B. Zhu X. Brown D. Birnbaumer L. J. Biol. Chem. 1998; 273: 8675-8679Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). These authors inferred the intracellular or extracellular location of both native and inserted glycosylation sites in the full-length TRPC3 sequence by transiently expressing appropriate recombinant proteins in COS cells and assaying for their glycosylation. They concluded that HR 1 was left out of the membrane on the cytosolic side and that HRs 2–6 and 8 were MSSs. A site placed near the N-terminal end of HR 7 was found to be glycosylated, consistent with its proposed role as a shallowly membrane-embedded part of the ion-conducting pore. They also verified that the N and C termini of TRPC3 were intracellular by showing that HA tags inserted at these sites were not accessible to extracellular antibodies unless the cells were permeabilized. However, analysis of the TRPC1 sequence using a number of recently derived algorithms for predicting MSSs as well as a detailed comparison of the TRPC1 and TRPC3 sequences suggested to us that a complementary study of the TRPC1 topology would be worth carrying out. For example, we found that HRs 1 and 7 of TRPC1 (the HR thought to be left out of the membrane in TRPC3 and the HR thought to form a part of the ion-conducting pore, respectively) were quite hydrophobic and almost universally predicted to be MSSs in TRPC1. We also noted that sequence conservation between TRPC1 and TRPC3 was rather poor within some of the HRs and their connecting loops (overall sequence identity over the eight HRs and interconnecting loops of TRPC1 and TRPC3 is ∼28%). The integration of membrane proteins into the bilayer of the ER has been shown to occur with the aid of a large complex of membrane-bound translocation/insertion machinery termed the "translocon" (6Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (501) Google Scholar, 7Turner R.J. J. Membr. Biol. 2003; 192: 149-157Crossref PubMed Scopus (7) Google Scholar). The functional core of this complex is a transmembrane aqueous channel sufficiently large to accommodate one or more MSS. Ribosomes that are synthesizing membrane proteins associate with the translocon in such a way that successive MSSs are fed into this channel where they are recognized and ultimately transferred laterally into the lipid bilayer in their proper transmembrane orientations (6Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (501) Google Scholar, 7Turner R.J. J. Membr. Biol. 2003; 192: 149-157Crossref PubMed Scopus (7) Google Scholar, 8Matlack K.E.S. Mothes W. Rapoport T.A. Cell. 1998; 92: 381-390Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 9Hegde R.S. Lingappa V.R. Cell. 1997; 91: 575-582Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In the simplest case, each MSS is sequentially recognized, oriented, and integrated into the membrane by the translocon as it is synthesized. But more complex scenarios where the integration of a MSS has been shown to depend on presence of neighboring MSSs or on the structure or charge of its flanking regions have been well documented (Ref. 7Turner R.J. J. Membr. Biol. 2003; 192: 149-157Crossref PubMed Scopus (7) Google Scholar and references therein). It also seems clear that multiple MSSs can occupy the translocon channel simultaneously and that some MSSs may exit the translocon into the bilayer en bloc (10Heinrich S.U. Mothes W. Brunner J. Rapoport T.A. Cell. 2000; 102: 233-244Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). In this paper, we examine the biogenesis and topology of TRPC1 in intact HEK-293T cells using a truncation mutant approach (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) that allows us to follow the integration/folding process described above. In contrast to results obtained with TRPC3, we find that HR 1 of TRPC1 spans the membrane consistent with its high hydrophobicity, and that HR 3 is left out of the membrane on the cytosolic side. In addition, our results suggest a mechanism by which a pore-like structure might form from HRs 6–8 during TRPC1 biogenesis. As discussed in more detail later in the paper, amino acids downstream of HR 8, including the highly conserved "TRP box" (Glu-Trp-Lys-Phe-Ala-Arg), appear to play an important role in this process. We suggest that this mechanism may be a common feature of the folding of the TRPC and related ion pores. DNA Constructs—Segments of the human TRPC1 sequence were cloned into the mammalian expression vector pEGFP-β whose construction has been described previously (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). This vector drives the expression of a fusion protein consisting of the enhanced green fluorescent protein (EGFP) followed by BglII and HindIII restriction sites for the insertion of additional sequence and finally a C-terminal glycosylation tag. The segments of TRPC1 indicated in the text and/or figure legends were amplified by PCR and cloned directly into pEGFP-β by standard methods. The forward and reverse PCR primers, incorporating 5′- and 3′-BglII and -HindIII sites, respectively, were designed essentially as reported in previous studies from our laboratory (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In all of the fusion protein constructs, the amino acids Ser-Asp-Leu and Gly-Ser-Phe coded (in part) by BglII and HindIII, respectively, flanked the TRPC1 inserts in pEGFP-β. All of the TRPC1 inserts began at M339. The correctness of all of the PCR products was confirmed by direct sequencing. In our early experiments, we were plagued by PCR errors in constructs extending beyond HR 4 (typically resulting in premature stop codons). This problem was resolved by growing all of the transformed bacteria at 30 °C rather than at 37 °C. We suspect that this problem was related to the production of a toxic protein that inhibited the growth of bacteria harboring correctly coded clones, but we have not explored this further. An HA-tagged human TRPC1 clone (in pcDNA3.1) was used as the template for the PCR reactions (12Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). For inserts longer than K647, the template was the same vector in which the native TRPC1 Hind III site following HR 8 had been destroyed by a silent mutation. Glycosylation consensus sites (see "Results") were inserted into the full-length HA-tagged TRPC1 sequence in pcDNA3.1 using the QuikChange kit (Stratagene) according to the manufacturer's instructions. Growth and Transfection of HEK-293T and HEK-293 Cells—HEK-293T cells (from American Type Culture Collection) were cultured in Dulbecco's modified essential medium supplemented with 2 mm glutamine, 100 μg/ml each of penicillin and streptomycin (all from Biofluids), and 10% heat-inactivated fetal bovine serum (Invitrogen). Cells were grown in 10-cm plastic dishes in a humidified incubator at 37 °C and 5% CO2 and subcultured every 2–3 days. Subconfluent (∼80%) HEK-293T monolayers were transiently transfected overnight (19–24 h) with the expression vectors indicated using Polyfect (Qiagen) according to the manufacturer's instructions. HEK-293 cells (from Microbix Biosystems Inc.) were cultured and transfected as above with the exception that Earle's minimal essential medium was used in place of Dulbecco's modified essential medium. To obtain stably transfected HEK-293 cells, G418 (0.9 mg/ml) was added to the medium 2 days after transfection and cells were subcultured as necessary. Confluent cultures of G418-resistant cells were harvested ∼3 weeks later. Preparation of Particulate and Membrane Fractions—Particulate and membrane fractions from HEK-293 and HEK-293T cells were obtained essentially as described previously (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Cells were washed in phosphate-buffered saline and then homogenized in ice-cold TEEA buffer consisting of 20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 3 mm EGTA, 300 μm AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride, ICN), 10 μm leupeptin, 10 μm pepstatin A, and 2.5 μg/ml aprotinin (all from Roche Applied Science). This material was centrifuged at 1,000 × g for 10 min, and the supernatant was saved. The pellet was resuspended in TEEA buffer, rehomogenized, and centrifuged as before. The combined supernates from these two homogenization steps were centrifuged at 100,000 × g for 30 min, and the resulting "particulate fraction" was resuspended in TEEA buffer, snap-frozen, and stored above liquid nitrogen (protein concentration was typically 5–10 mg/ml measured using the Bio-Rad protein assay kit with bovine IgG as the standard). The "membrane fraction" was prepared from the above particulate fraction by an alkaline floatation step (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 13Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Crossref PubMed Scopus (263) Google Scholar, 14Ota K. Sakaguchi M. von Heijne G. Hamasaki N. Mihara K. Mol. Cell. 1998; 2: 495-503Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) as follows. An aliquot of the particulate fraction containing 50–100 μg of protein was diluted to 25 μl with TEEA, and 25 μl of 200 mm Na2CO3 (pH 12.0) was added. This mixture was incubated on ice for 30 min and then mixed with 90 μl of 2.5 m sucrose in 100 mm Na2CO3. 50 μl of 1.25 m sucrose and 50 μl of 0.25 m sucrose, both containing 0.2 mm EDTA and 10 mm Tris-HCl (pH 8.0), were overlaid next on the alkaline mixture, and the tube was centrifuged at 100,000 × g for 60 min in a Beckman TL100 ultracentrifuge equipped with a TLA100.3 rotor. The 0.25 and 1.25 m sucrose layers and the interface between the 1.25 m sucrose layer and the alkaline mixture were recovered as the membrane fraction. Deglycosylation of the Membrane Fraction—Aliquots of the above membrane fractions were treated with peptide N-glycosidase F (PNGase F, New England Biolabs) as follows. A 10-μl aliquot of the membrane fraction was diluted to 20 μl in 50 mm sodium phosphate (pH 7.5), 0.5% SDS, and 1% β-mercaptoethanol (final concentrations) and incubated at room temperature for 10 min. 2.22 μl of 10% Nonidet P-40 and 1 μl (1,000 units) of PNGase F next were added, and this mixture was incubated at 37 °C for 2 h. In control samples, PNGase F was substituted by its storage buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm Na2EDTA, 50% glycerol). Western Blotting and Analysis—SDS-PAGE using 4–20% Tris-glycine Ready gels (Bio-Rad) and Western blotting using a rabbit antigreen fluorescent protein polyclonal antibody (Molecular Probes) were carried out as described previously (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). SDS-PAGE using 10% Tris-glycine Ready Gels (Bio-Rad) and Western blotting using a peroxidase-conjugated anti-HA antibody (Roche Applied Science) were carried out similarly with the exception that incubation with the primary antibody was for 90 min and no secondary antibody was used. Quantitation of Western blots was done using a Molecular Dynamics computing densitometer. Quantitative results shown are means ± S.E. for three or more independent experiments. Predictions of Membrane-spanning Segments—The MSSs of human TRPC1 predicted by a number of recent theoretical methods are summarized in the upper portion of Fig. 1. In each case, the positions of predicted MSSs in the amino acid sequence are indicated by horizontal lines. The results illustrated are from the DAS (15Cserzo M. Wallin E. Simon I. von Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar), SOSUI (16Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics. 1998; 14: 378-379Crossref PubMed Scopus (1577) Google Scholar), PRED-TMR (17Pasquier C. Promponas V.J. Palaios G.A. Hamodrakas J.S. Hamodrakas S.J. Protein Eng. 1999; 12: 381-385Crossref PubMed Scopus (145) Google Scholar), Toppred2 (18Claros M.G. von Heijne G. Comput. Appl. Biosci. 1994; 10: 685-686PubMed Google Scholar), the PHDhtm (19Rost B. Fariselli P. Casadio R. Protein Sci. 1996; 5: 1704-1718Crossref PubMed Scopus (533) Google Scholar), TMHMM (20Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9290) Google Scholar), and HMMTOP (21Tusnady G.E. Simon I. Bioinformatics. 2001; 17: 849-850Crossref PubMed Scopus (1580) Google Scholar) methods. All of these methods have been shown to be considerably more accurate in the prediction of MSSs than a simple hydropathy analysis (22Chen C.P. Kernytsky A. Rost B. Protein Sci. 2002; 11: 2774-2791Crossref PubMed Scopus (176) Google Scholar), and all are available for use over the Internet (see Refs. 15Cserzo M. Wallin E. Simon I. von Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar, 16Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics. 1998; 14: 378-379Crossref PubMed Scopus (1577) Google Scholar, 17Pasquier C. Promponas V.J. Palaios G.A. Hamodrakas J.S. Hamodrakas S.J. Protein Eng. 1999; 12: 381-385Crossref PubMed Scopus (145) Google Scholar, 18Claros M.G. von Heijne G. Comput. Appl. Biosci. 1994; 10: 685-686PubMed Google Scholar, 19Rost B. Fariselli P. Casadio R. Protein Sci. 1996; 5: 1704-1718Crossref PubMed Scopus (533) Google Scholar, 20Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9290) Google Scholar, 21Tusnady G.E. Simon I. Bioinformatics. 2001; 17: 849-850Crossref PubMed Scopus (1580) Google Scholar). In the lower part of Fig. 1, we show a hydrophobicity plot obtained by the classical method of Kyte and Doolittle (23Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17297) Google Scholar) using a 19-amino acid window. The eight HRs are labeled. Note that all of the theoretical methods predict that HR 1 is a MSS, and most predict that HR 7 is membrane spanning but that HR 3 is not. There is also some disagreement among the methods concerning the integration and location of HRs 4 and 5. The amino acid sequence of the central hydrophobic domain of TRPC1 with the approximate positions of HRs 1–8 indicated is shown in Fig. 2.Fig. 2Amino acid sequence of human TRPC1. The "approximate" positions of HRs 1 through 8 are underlined. The amino acids marked with asterisks indicate the truncation points of the TRPC1 inserts in pEGFP-β described in the paper. The amino acids marked with pound signs are the mutated residues in the experiment shown in Fig. 4A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Experimental System—To explore the topology of TRPC1, we have used a truncation mutant approach (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) where portions of the TRPC1 sequence beginning at Met-339 and containing one or more possible MSSs are fused (see "Materials and Methods") between EGFP and the extracellular tail (177 amino acids) of the β-subunit of the rabbit gastric H,K-ATPase, a glycosylation tag. This latter sequence contains five consensus sites for N-linked glycosylation (24Bamberg K. Sachs G. J. Biol. Chem. 1994; 269: 16909-16919Abstract Full Text PDF PubMed Google Scholar). When translocated into the interior of the ER, it acquires ∼14 kDa of apparent molecular weight due to glycosylation (25Gerelsaikhan T. Turner R.J. J. Biol. Chem. 2000; 275: 40471-40477Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), an increase that is easily detected by SDS-PAGE electrophoresis. The use of this glycosylation tag in membrane topology determinations is now well established (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 24Bamberg K. Sachs G. J. Biol. Chem. 1994; 269: 16909-16919Abstract Full Text PDF PubMed Google Scholar, 25Gerelsaikhan T. Turner R.J. J. Biol. Chem. 2000; 275: 40471-40477Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 26Bayle D. Weeks D. Sachs G. J. Biol. Chem. 1995; 270: 25678-25684Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27Bayle D. Weeks D. Sachs G. J. Biol. Chem. 1997; 272: 19697-19707Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). EGFP at the N terminus of our constructs acts as both a cytosolic anchor and a convenient marker for the detection of fusion proteins on Western blots. The Membrane Topology of HRs 1–3—In Fig. 3A, we show the results of a series of experiments where TRPC1 fragments including HR 1 (K373), HRs 1 and 2 (M416), and HRs 1, 2, and 3 (W443 and Q457) were expressed as EGFP/β-subunit fusion proteins in HEK-293T cells. In each of the panels in Fig. 3A, we show the results of a typical experiment where the membrane fraction from HEK-293T cells, transiently transfected with the truncation mutant indicated, was treated with (+) or without (–) PNGase F (see "Materials and Methods"). These membrane fractions were run on SDS-PAGE and probed by Western blotting to determine the extent of glycosylation of the β-subunit and thus its location inside or outside the ER lumen. The percentage of glycosylated recombinant protein is given below each panel. Thus, for example, the mutant truncated after HR 1 (K373) is highly glycosylated (∼90%), indicating that HR 1 crosses the ER membrane in a Ncyt/Clum orientation consistent with its high hydrophobicity and with the theoretical predictions discussed above (cf. Fig. 1). As detailed under "Materials and Methods," the membrane preparations analyzed in Fig. 3A were obtained from particulate fractions of HEK-293T cells using an alkaline floatation procedure (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 13Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Crossref PubMed Scopus (263) Google Scholar, 14Ota K. Sakaguchi M. von Heijne G. Hamasaki N. Mihara K. Mol. Cell. 1998; 2: 495-503Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In this procedure, the particulate fractions were first incubated in alkaline 100 mm Na2CO3. Under these conditions, any membrane vesicles present are converted to sheets and most protein-protein interactions are disrupted; however, protein-lipid (hydrophobic) interactions are not disrupted and the membrane bilayer remains intact (28Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1385) Google Scholar). Thus, this incubation is expected to strip away most peripheral membrane proteins but leave integrated proteins in the bilayer. Following this incubation in alkaline medium, the membrane fraction was isolated by floatation on a sucrose gradient (see "Materials and Methods") (11Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 13Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Crossref PubMed Scopus (263) Google Scholar, 14Ota K. Sakaguchi M. von Heijne G. Hamasaki N. Mihara K. Mol. Cell. 1998; 2: 495-503Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In addition to separating the membranes from the peripheral proteins removed by alkaline treatment, this step also leaves any aggregated recombinant proteins arising from overexpression in the lower phase. An analysis of the particulate and membrane fractions from cells expressing K373 showed that 73 ± 3% of this recombinant protein was glycosylated in the particulate fraction and that 24 ± 8% of the total K373 found in the particulate fraction was recovered in the membrane fraction (data not shown). From these results, one can calculate that ∼70% of the glycosylated K373 found in the particulate fraction does not copurify with the membrane fraction. Because glycosylation can only occur in the lumen of the ER, these non-membrane-associated recombinant proteins must have been formerly inserted into the membrane and then later removed, presumably for degradation. Also, because membrane proteins extracted from the ER and targeted for degradation are first deglycosylated (29Tsai B. Ye Y. Rapoport T.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 246-255Crossref PubMed Scopus (552) Google Scholar), the percentage of glycosylated K373 in the particulate fraction (73 ± 3%) is expected to be a lower limit on the percentage of these recombinant proteins targeted to the membrane. Returning to Fig. 3A, we see that the mutant truncated after HR 2 (M416) is ∼60% glycosylated, indicating that HR 2 crosses the membrane in a Nlum/Ccyt orientation in only 40% of these recombinant proteins. Extending the length of the TRPC1 insert to W430 to include the N-terminal end of HR 3 (Fig. 2) has no significant effect on this result (data not shown); however, when the TRPC1 sequence was extended to include all of HR 3 (W443), almost no glycosylation was observed, indicating that the C terminus of this truncation mutant is mainly cytosolic. Further extension of the TRPC1 sequence to Gln-457 to include the sequence between HR 3 and HR 4 yielded a similar result (Fig. 3A). Taken together, these results indicate that HR 1 is a MSS with a Ncyt/Clum orientation and that a second MSS with a Nlum/Ccyt orientation, presuma
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