US2, a Human Cytomegalovirus-encoded Type I Membrane Protein, Contains a Non-cleavable Amino-terminal Signal Peptide
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m107904200
ISSN1083-351X
AutoresBenjamin E. Gewurz, Hidde L. Ploegh, Domenico Tortorella,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoThe human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH2 terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH2-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable −3,−1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein. The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH2 terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH2-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable −3,−1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein. Signal peptides dictate ER insertion of integral membrane proteins and proteins destined for the secretory pathway in either co- or post-translational fashion (1.Martoglio B. Dobberstein B. Trends Cell Biol. 1998; 8: 410-415Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 2.von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (843) Google Scholar, 3.von Heijne G. Nature. 1998; 396: 111-113Crossref PubMed Scopus (99) Google Scholar). Signal peptides are recognized by the signal recognition particle, which directs the nascent chain and ribosome to the signal recognition particle receptor embedded within ER 1The abbreviations used are: ERendoplasmic reticulumHCMVhuman cytomegalovirusEndoHendoglycosidase HHCclass I heavy chainFGFfibroblast growth factoraaamino acid(s)β2mβ2-microglobulin 1The abbreviations used are: ERendoplasmic reticulumHCMVhuman cytomegalovirusEndoHendoglycosidase HHCclass I heavy chainFGFfibroblast growth factoraaamino acid(s)β2mβ2-microglobulin membrane (4.Hegde R.S. Lingappa V.R. Trends Cell Biol. 1999; 9: 132-137Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 5.Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (496) Google Scholar, 6.Keenan R.J. Freymann D.M. Walter P. Stroud R.M. Cell. 1998; 94: 181-191Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 7.Rapoport T.A. Jungnickel B. Kutay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (491) Google Scholar, 8.Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (708) Google Scholar, 9.Zheng T. Nicchitta C.V. J. Biol. Chem. 1999; 274: 36623-36630Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Upon docking with the ER membrane, many proteins are co-translationally inserted into the ER lumen via a ribosome-translocon channel that includes the heterotrimeric Sec61p complex and the translocating chain-associating membrane protein (10.Jungnickel B. Rapoport T.A. Cell. 1995; 82: 261-270Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 11.Voigt S. Jungnickel B. Hartmann E. Rapoport T.A. J. Cell Biol. 1996; 134: 25-35Crossref PubMed Scopus (151) Google Scholar). Once the translocon has been engaged, NH2-terminal signal peptides are cleaved from the nascent chain by signal peptidase, a serine endopeptidase present near the translocon in the ER membrane (12.Dalbey R.E. Von Heijne G. Trends Biochem. Sci. 1992; 17: 474-478Abstract Full Text PDF PubMed Scopus (177) Google Scholar). Signal-anchor sequences also interact transiently with the ER translocation complex, but are not cleaved. Instead, signal-anchor sequences move laterally out of the translocon to become permanent membrane anchors (13.Heinrich S.U. Mothes W. Brunner J. Rapoport T.A. Cell. 2000; 102: 233-244Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 14.Nilsson I. Whitley P. von Heijne G. J. Cell Biol. 1994; 126: 1127-1132Crossref PubMed Scopus (124) Google Scholar). endoplasmic reticulum human cytomegalovirus endoglycosidase H class I heavy chain fibroblast growth factor amino acid(s) β2-microglobulin endoplasmic reticulum human cytomegalovirus endoglycosidase H class I heavy chain fibroblast growth factor amino acid(s) β2-microglobulin Signal peptides are composed of three distinct regions, n-, h-, and c-regions. The n-region consists of polar residues, often with a net positive charge, at the NH2 terminus (15.von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1516) Google Scholar). The h-region is the central hydrophobic 7–15 residue helical core that can insert into the ER membrane (13.Heinrich S.U. Mothes W. Brunner J. Rapoport T.A. Cell. 2000; 102: 233-244Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 16.Plath K. Mothes W. Wilkinson B.M. Stirling C.J. Rapoport T.A. Cell. 1998; 94: 795-807Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Finally, the carboxyl-terminal c-region has more polar character and contains the signal peptidase cleavage site (15.von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1516) Google Scholar). Signal peptidase recognizes a pattern that includes amino acids with small side chains in the −1 and −3 positions relative to the cleavage site, present in the c-region in extended conformation near the head groups on the inner leaflet of the ER membrane (2.von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (843) Google Scholar, 14.Nilsson I. Whitley P. von Heijne G. J. Cell Biol. 1994; 126: 1127-1132Crossref PubMed Scopus (124) Google Scholar, 16.Plath K. Mothes W. Wilkinson B.M. Stirling C.J. Rapoport T.A. Cell. 1998; 94: 795-807Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar,17.von Heijne G. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1596) Google Scholar). The human cytomegalovirus (HCMV) encodes two ER-resident membrane glycoproteins, US2 and US11, that each destabilize major histocompatibility class I molecules (18.Tortorella D. Gewurz B.E. Furman M.H. Schust D.J. Ploegh H.L. Annu. Rev. Immunol. 2000; 18: 861-926Crossref PubMed Scopus (705) Google Scholar). The 199-residue US2 glycoprotein contains an ER-lumenal portion, a predicted single transmembrane domain, and a short cytoplasmic tail (Fig. 1). US2 recognizes class I molecules via an immunoglobulin-like fold that attaches to the class I ER-lumenal domain (19.Gewurz B.E. Gaudet R. Tortorella D. Wang E.W. Ploegh H.L. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6794-6799Crossref PubMed Scopus (128) Google Scholar) and US2 subsequently targets class I heavy chains for dislocation from the ER to the cytosol, where they are rapidly degraded by the proteasome (20.Wiertz E.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (948) Google Scholar). The mechanism of ER dislocation and degradation is not clearly understood. HCMV US2 and US11 exhibit unusual characteristics concerning their ER processing events. The US11 signal peptide is cleaved from the nascent chain in delayed fashion (21.Rehm A. Stern P. Ploegh H.L. Tortorella D. EMBO J. 2001; 20: 1573-1582Crossref PubMed Scopus (55) Google Scholar). Both the signal peptide n-region and the COOH-terminal membrane anchor influence processing of the US11 signal peptide (21.Rehm A. Stern P. Ploegh H.L. Tortorella D. EMBO J. 2001; 20: 1573-1582Crossref PubMed Scopus (55) Google Scholar). Here we report several highly unusual properties of US2 ER translocation. We show that US2 behaves like a type I membrane protein that contains a non-cleavable signal sequence at its NH2 terminus. U373-MG astrocytoma cells stably transfected with US21–199 cDNA (22.Jones T.R. Hanson L.K. Sun L. Slater J.S. Stenberg R.M. Campbell A.E. J. Virol. 1995; 69: 4830-4841Crossref PubMed Google Scholar) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 5% calf serum, and 0.375 μg/ml puromycin. All variants of US2 were stably transfected into U373 cells and maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 5% calf serum, and 0.5 mg/ml geneticin (Invitrogen, Frederick, MD). The human embryonic kidney cell line (HEK-293) was maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 5% calf serum. Polyclonal antisera generated against US2 and class I heavy chains were generated as described (23.Tortorella D. Story C.M. Huppa J.B. Wiertz E.J. Jones T.R. Bacik I. Bennink J.R. Yewdell J.W. Ploegh H.L. J. Cell Biol. 1998; 142: 365-376Crossref PubMed Scopus (113) Google Scholar). The anti-β2m serum was generated as described (21.Rehm A. Stern P. Ploegh H.L. Tortorella D. EMBO J. 2001; 20: 1573-1582Crossref PubMed Scopus (55) Google Scholar). The cDNA of full-length US2 (amino acids 1–199) was cloned from the AD169 HCMV genome into the eukaryotic expression vector pCDNA 3.1 (Invitrogen, Carlsbad, CA). US2 truncation mutants, US220–199 (aa 20–199) and US21–160 (aa 1–160) were subcloned from US2 (pCDNA 3.1) by PCR and inserted into pcDN3.1. Major histocompatibility class I (HLA-A2) heavy chain lacking a signal sequence (HC25–365) was subcloned from HLA-A2 (pcDNA3.1) by PCR and inserted into pcDNA3.1. The H-2Kb/US2 chimeras, Kb1–5/US27–199 (H-2Kb(aa 1–5)/US2 (aa 7–199)) and Kb1–16/US217–199((H-2Kb (aa 1–16)/US2 (aa 17–199)), and US2/HLA-A2 chimeras, US21–20/HC25–365 ((US2 (aa 1–20)/HLA-A2 (aa 25–365)), US21–25/HC25-365 (US2 (aa 1–25)/HLA-A2 (aa 25–365)), US21–30/HC25–365 (US2 (aa 1–30)/HLA-A2 (aa 25–365)), US21–35/HC25–365 (US2 (aa 1–35)/HLA-A2 (aa 25–365)), US21–40/HC25–365(US2 (aa 1–40)/HLA-A2 (aa 25–365)), were generated by amplifying the appropriate fragment by PCR followed by ligation of two of the respective fragments. Using specific primers to the sense and antisense strand of the ligated product, the chimeric cDNAs were amplified by PCR, phosphorylated with T4 DNA kinase, and inserted into pCDNA 3.1. The H-2Kb/US2 chimeric mutant, Kb1–21/US221–199(H-2Kb(aa 1–21)/US2(aa 21–199)) was generated by amplifying the US2 cDNA that corresponds to aa 21–199 by PCR. This truncated US2 was ligated to the murine H2 class I heavy chain Kb signal sequence (H-2Kb residues 1–21) (pSP72 (Invitrogen)) (24.Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (79) Google Scholar). The Kb1–21/US221–199 cDNA was then shuttled into pCDNA 3.1. Lipid-mediated transient (HEK-293 cells) and stable (U373-MG cells) transfections were performed as described by the manufacturer (5 μg of DNA/15 μl of LipofectAMINE (Invitrogen) were used per transfection). Cells were released from tissue culture flasks by trypsinization and incubated in cysteine/methionine-free Dulbecco's modified Eagle's medium for 45 min at 37 °C. Cells were metabolically labeled with 500 μCi of [35S]cysteine/methionine (1200 Ci/mmol; PerkinElmer Life Sciences, Boston, MA) at 37 oC for the indicated times. Pulse-chase experiments and immunoprecipitation were performed as previously described (21.Rehm A. Stern P. Ploegh H.L. Tortorella D. EMBO J. 2001; 20: 1573-1582Crossref PubMed Scopus (55) Google Scholar). Digestion with endoglycosidase H (EndoH) (New England Biolabs, Beverly, MA) was performed on immunoprecipitated complexes according to the manufacturer's instructions. Proteins were separated by SDS-PAGE and [35S]methionine-labeled proteins were visualized by fluorography/autoradiography (25.Ploegh H.L. Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. Wiley, New York1995Google Scholar). In vitrotranscription and translation of US21–199 were performed as described (26.Furman M.H. Ploegh H.L. Tortorella D. J. Biol. Chem. 2002; 277: 3258-3267Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The subcellular fractionation and Na2CO3 treatment were performed as previously described, respectively (21.Rehm A. Stern P. Ploegh H.L. Tortorella D. EMBO J. 2001; 20: 1573-1582Crossref PubMed Scopus (55) Google Scholar). Urea treatment of homogenates prepared from HEK-293 transfectants was performed in a similar manner as Na2CO3 treatment, except 4.5 m urea was used. Following SDS-gel electrophoresis, US2 was transferred from the polyacrylamide gel to a Sequi-Blot polyvinylidene difluoride membrane (0.2 mm) (Bio-Rad, Hercules, CA) using a semi-dry blotting apparatus (Labconco, Seattle, WA). The glycosylated US2 polypeptide was excised from the polyvinylidene difluoride membrane and subjected to automated Edman degradation. An Applied Biosystem Protein Sequencer, Model 477, using ATZ chemistry, at the Biopolymers Laboratory at MIT, Center for Cancer Research was utilized. The fractions from each degradation cycle were collected and counted by liquid scintillation spectrometry. The nonpolar region present at the US2 COOH terminus likely constitutes a membrane anchor (Fig. 1), consistent with earlier proposals for the topology of US21–199 (19.Gewurz B.E. Gaudet R. Tortorella D. Wang E.W. Ploegh H.L. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6794-6799Crossref PubMed Scopus (128) Google Scholar, 20.Wiertz E.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (948) Google Scholar). To establish more directly the membrane disposition of US2, Na2CO3 extraction was performed on homogenates of metabolically labeled ([35S]methionine) US21–199 cells (see "Experimental Procedures"). Treatment of cellular homogenates with Na2CO3releases soluble and peripheral membrane proteins, while transmembrane proteins remain inserted in the lipid bilayer. The membrane pellet (P) and soluble (S) fractions from Na2CO3-treated homogenates were separated by centrifugation (150,000 ×g), followed by recovery of either US21–199(Fig. 2A, lanes 1–3) or the soluble protein β2-microglobulin (β2m) (Fig. 2A, lanes 4–6) by anti-US2 and anti-β2m serum, respectively. Whereas N-linked glycosylated US21–199 molecules were recovered exclusively from the insoluble, sedimentable fraction (Fig. 2A, lane 3), β2m molecules were present only in the soluble fraction (Fig. 2A, lane 5). We conclude that US21–199 is a membrane protein. N-Linked glycosylation was examined to further assess the topology of US21–199. It contains consensus N-linked glycan attachment sequons at residues 68–70, 172–174, and 188–190 (Fig. 1); the latter two are within the predicted transmembrane anchor and cytoplasmic tail, respectively. To determine the number of glycosylation sites utilized in vivo, US2 immunoprecipitates were treated with varying amounts of EndoH. Independently of enzyme concentration, EndoH treatment produces a single novel polypeptide, whose mass is reduced by ∼4 kDa, consistent with the presence of a single N-linked glycan on US2 (Fig. 2B). Furthermore, US2 deletion mutants that lack the two COOH-terminal glycosylation sequons are glycosylated in a similar manner in transfectants (Fig. 5). We conclude that a single N-linked glycan is attached at asparagine 68, present in a loop between the B and C β-strands of the US2 Ig-like fold (Fig. 6A) (19.Gewurz B.E. Gaudet R. Tortorella D. Wang E.W. Ploegh H.L. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6794-6799Crossref PubMed Scopus (128) Google Scholar).Figure 6US21–160 is a soluble protein. A, the hydrophobic stretch between residues 110–130 of US2 is located in the ER lumen; the US2·HLA-A2·Tax complex structure (19.Gewurz B.E. Gaudet R. Tortorella D. Wang E.W. Ploegh H.L. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6794-6799Crossref PubMed Scopus (128) Google Scholar): class I heavy chain (yellow), β2-microglobin (gray), US2 (blue, residues 43–109 and 131–137;red, residues 110–130), and Tax peptide (green). B and C, homogenates from metabolically labeled HEK-293 cells transfected with US21–199, US21–160, and β2m were treated with 100 mm Na2CO3(B) or 4.5 m (C) urea followed by high speed centrifugation (150,000 × g) (150Kg). US21–199 (lanes 1–3), US21–160 (lanes 4–6), and β2m (lanes 7–9) were recovered directly from cell lysates (−) (lanes 1, 4, and 7), from the 100 mm Na2CO3 or 4.5m 150,000 × g supernatant (S) (lanes 2, 5, and 8) and from the 100 mmNa2CO3 or 4.5 m urea 150,000 × K pellet (P) (lanes 3, 6, and 9), respectively, using the respective sera and analyzed by SDS-PAGE (15%). CHO, carbohydrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) US2 polypeptides that differ by the presence of the single N-linked glycan are recovered from lysates of metabolically labeled US21–199 cells (Fig. 2B). To determine whether the non-glycosylated species arises as a deglycosylated degradation intermediate produced in the course of a dislocation reaction (20.Wiertz E.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (948) Google Scholar), pulse-chase experiments were performed on US21–199 cells in the presence and absence of the proteasome inhibitor, ZL3VS (27.Bogyo M. McMaster J.S. Gaczynska M. Tortorella D. Goldberg A.L. Ploegh H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6629-6634Crossref PubMed Scopus (405) Google Scholar). US2 molecules were recovered from cell lysates and analyzed by SDS-PAGE (Fig. 3A). The recovery of glycosylated and non-glycosylated US2 polypeptides decreases at the later chase times both in the presence or absence of ZL3VS (Fig. 3A), but as expected, the degradation rate of non-glycosylated US21–199 is slower in the presence of proteasome inhibition (Fig. 3, compare lanes 1–4 and 5–8). We observed no evidence for a precursor-product relationship for glycosylated and non-glycosylated US2 polypeptides. We infer that the non-glycosylated US2 polypeptide does not arise as a degradation intermediate. Non-glycosylated US2 could result either from incomplete ER insertion or inefficient glycosylation. To distinguish between these possibilities, the localization of non-glycosylated US2 was determined by subcellular fractionation. Metabolically labeled US21–199 cells were homogenized by mechanical disruption, followed by differential centrifugation (Fig. 3B). The 100,000 × g supernatant (S) contains cytosolic proteins, while the 100,000 × g pellet (P) contains the microsomal fraction. Glycosylated US2 is recovered exclusively from the 100,000 × g pellet (P) (Fig. 3B, lane 4), indicative of its membrane disposition. However, the bulk of the non-glycosylated US2 polypeptide is recovered from the cytosolic fraction, with only a minor fraction co-sedimenting with the microsomal fraction (Fig. 3B, lanes 3 and 4). The cytosolic form of US2 is probably material that did not insert into the ER. The interaction of US2 with a membrane protein, possibly through its signal peptide, may account for the association of non-glycosylated US2 with the microsomal fraction. A subpopulation of non-glycosylated US2 continues to co-sediment with the microsomal fraction upon Na2CO3extraction. 2B. E. Gewurz, H. L. Ploegh, and D. Tortorella, unpublished data. These results fail to resolve completely the source of non-glycosylated US2. However, in the absence of a precursor-product relationship for glycosylated and non-glycosylated US2 in the presence and absence of proteasome inhibition, we favor the notion that membrane insertion, and hence N-linked glycosylation, did not occur for non-glycosylated US2. The SignalP computer algorithm (www.cbs.dtu.dk/services/SignalP/index.html) predicts a US2 NH2-terminal signal peptide with probable cleavage site between residues 20 and 21. However, most signal peptides exhibit greater hydrophobicity than that present at the US2 NH2terminus (Fig. 1) (14.Nilsson I. Whitley P. von Heijne G. J. Cell Biol. 1994; 126: 1127-1132Crossref PubMed Scopus (124) Google Scholar). We therefore examined whether the US2 NH2 terminus corresponds to a bona fide signal peptide. An NH2-terminal truncation mutant that lacks the NH2-terminal 20 residues (US220–199) was stably transfected into U373 cells and examined for ER insertion by the acquisition of the N-linked glycan. US2 molecules were recovered from SDS-treated lysates of metabolically labeled US21–199 and US220–199 cells using anti-US2 serum. Half of the US2 immunoprecipitates were treated with EndoH and analyzed by SDS-PAGE (Fig. 4A). In contrast to US21–199, the US220–199 polypeptides recovered from cell lysates were not glycosylated, as judged from their resistance to EndoH, and were of a size consistent with that of the predicted non-glycosylated polypeptide (Fig. 4A, lanes 3 and 4). Subcellular fractionation likewise indicated that US220–199 molecules localize to the cytosol (Fig. 4B). Nearly all of the US220–199 molecules were recovered from the 100,000 × g supernatant (S) fraction (Fig. 4B, lane 3). In contrast, a major population of the US2 chimera that contains the signal peptide of murine H-2Kb as the replacement for residues 1–20 of US2 (Kb1–21/US221–199) was inserted into the ER (Fig. 4A, lanes 5 and 6). Not only does the 21-residue signal sequence of murine H-2Kb class I heavy chain drive the ER insertion of US220–199 (as evidenced by EndoH sensitivity of the Kb1–21/US221–199 chimera (Fig. 4A, lanes 5 and 6)), the glycosylated Kb1–21/US221–199 species (Fig. 4A, lane 5, upper band) also has a faster electrophoretic mobility than glycosylated US21–199(Fig. 4A, compare lanes 1 and 5). This difference in mobility between US21–199 and Kb1–21/US221–199 is also observed when the N-linked glycans are removed by EndoH treatment (Fig. 4A, compare lanes 2 and 6). This result was altogether unexpected and suggests that the US21–199 molecule fully retains its NH2-terminal signal peptide. Consistent with this possibility, US2 translated in vitro in the absence of microsomal membranes (Fig. 4C, lane 1) co-migrates with EndoH-treated US2 molecules translated in the presence of microsomes (Fig. 4C, lane 3). To confirm the absence of signal peptide cleavage for US2, NH2-terminal sequencing was carried out on glycosylated US2 molecules recovered from metabolically labeled cellular transfectants (Fig. 4D). The major peak of radioactivity is observed at position one, with a smaller amount of radioactivity released at position 15, consistent with methionines present in the US2 sequence at residues 1 and 15 (Fig. 1). We conclude that US2 fully retains its NH2 terminus following ER insertion. A type I membrane protein that lacks an NH2-terminal signal sequence is without precedent. Internal hydrophobic domains can serve as internal signal peptides as well as stop transfer sequences. Such hydrophobic domains are observed in most proteins that lack a cleavable signal sequence. Is it possible that the putative transmembrane domain (Fig. 1, aa 161–185) of US21–199 can act as a signal peptide? We constructed a US2 COOH-terminal deletion mutant (US21–160) and examined its ability to insert into ER. We used glycosylation of US21–160 as a marker for ER insertion. U373 cells that stably express US21–160 still show efficient glycosylation of US21–160 (Fig. 5). The elimination of the putative transmembrane segment does not interfere with ER insertion of US2 and therefore the US2 contains an NH2-terminal signal peptide. A Kyte-Doolittle hydropathy plot of US21–199 demonstrates hydrophobic segments at the NH2 terminus (residues 1–20) and between residues 110 and 130, in addition to the putative transmembrane domain (Fig. 1). Can these hydrophobic domains act as additional membrane anchors? The crystal structure of the US2/class I complex shows that residues 110–130 comprise the F and G β-strands of the US2 Ig-like fold, indeed forming an important part of the class I binding surface (Fig. 6A) (19.Gewurz B.E. Gaudet R. Tortorella D. Wang E.W. Ploegh H.L. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6794-6799Crossref PubMed Scopus (128) Google Scholar). Therefore, this hydrophobic segment resides within the ER lumen. Consistent with this data, an additional N-linked glycosylation sequon introduced at position 149 is efficiently utilized (supplemental Fig. 1). Since this US2 mutant acquires N-linked glycans at positions 68 and 149, the intervening segment must also be present in the ER. Together, these results demonstrate that US2 residues 110–130 do not traverse the membrane bilayer and are consistent with a type I topology for US2. We next examined the cellular disposition of US21–160 to determine whether the uncleaved NH2-terminal signal peptide of US2 is a membrane anchor. US21–199, US21–160, and β2m were recovered from the 150,000 × g membrane pellet (P) and soluble (S) fractions of Na2CO3-treated homogenates of HEK-293-transfected cells (Fig. 6B). The membrane protein US21–199 (Fig. 6B, lanes 1–3) was recovered exclusively from the membrane fraction (P) of Na2CO3-treated homogenates (Fig. 6B, lane 3). In contrast, the soluble protein β2m (Fig. 6B, lanes 7–9) was recovered exclusively from the soluble fraction (S) of Na2CO3-treated homogenates (Fig. 6B, lane 8). In Na2CO3-treated homogenates, US21–160 is mostly recovered from the pellet fraction (P) (Fig. 6B, lane 6), while a small population of US21–160 molecules was recovered from the soluble fraction (S) (Fig. 6B, lane 5). Since protein-protein interactions can survive Na2CO3 treatment (28.Soriano S. Thomas S. High S. Griffiths G. D'Santos C. Cullen P. Banting G. Biochem. J. 1997; 324: 579-589Crossref PubMed Scopus (38) Google Scholar,29.Miller J.D. Tajima S. Lauffer L. Walter P. J. Cell Biol. 1995; 128: 273-282Crossref PubMed Scopus (89) Google Scholar) and could allow US2 to partition with the pellet fraction, US21–160 homogenates were treated with the weak denaturant 4.5 m urea (Fig. 6C) as an alternative to Na2CO3 treatment. Nearly all of the US21–160 molecules were recovered from the soluble (S) fraction upon urea treatment (Fig. 6C, lane 5). These results suggest that protein-protein interactions tether US21–160 to the ER membrane and that US21–160is itself a soluble protein. Note that the membrane protein US21–199 (Fig. 6C, lanes 1–3) is not released even to the slightest extent into the soluble fraction upon urea treatment (Fig. 6C, lane 2). The SignalP algorithm was used to predict the n-, h-, and c-regions of both the putative US2 and H-2Kb signal peptides. To determine which region of US2's signal peptide is responsible for the observed lack of signal peptide cleavage, we replaced the predicted US2 n, n + h, or n + h + c regions with the corresponding regions of the H-2Kbsignal peptide (Fig. 7A). US2 chimeras that contain the Kb n-region in place of the putative US2 n-region (Kb1–5/US27–199) are poorly inserted in the ER within these transfectants (Fig. 7B, lanes 3 and 4). Chimeras that contain the putative n + h-region of H-2Kb(Kb1–16/US217–199) are efficiently glycosylated (Fig. 7B, lanes 5 and 6). Despite efficient ER translocation and glycosylation, the hybrid Kb1–16/US217–199signal peptide apparently remains uncleaved following ER insertion (Fig. 7B, lanes 5 and 6). In contrast, chimeras that contain the entire H-2Kb signal sequence in place of the US2 20 NH2-terminal residues (Kb1–21/US221–199) are efficiently inserted into the ER with concomitant signal peptide cleavage (Fig. 7B, lanes 7 and 8). For the Kb1–21/US221–199 chimera, its signal peptide appears to be removed, since this product migrates faster than US21–199 and other H-2Kb/US2 chimeras upon SDS-PAGE separation. Thus, elements within the putative US2 signal peptide c-region are responsible for the lack of signal peptide cleavage. To determine whether the US2 NH2 terminus can direct an exogenous membrane protein to the ER, we constructed chimeras comprised of US2 NH2-terminal peptides fused to a reporter type I membrane protein, the major histocompatibility class I (HLA-A2) heavy chain (HC), lacking its own signal peptide (Fig. 8). Since the length of the US2 signal peptide is not precisely known, we generated a series of chimeric proteins that contain either the NH2-terminal 20, 25, 30, 35, or 40 residues of US2 fused to the NH2-ter
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