Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.022590
ISSN1083-351X
AutoresCatherine A. Bue, Charles Barlowe,
Tópico(s)Microtubule and mitosis dynamics
ResumoEfficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport. Efficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport. Anterograde transport in the eukaryotic secretory pathway is initiated by the formation of COPII 2The abbreviations used are: COPIIcoat protein complex IIALPalkaline phosphataseCOPIcoat protein complex IERendoplasmic reticulumwtwild typeHAhemagglutininDSPdithiobissuccinimidyl propionateSNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptor. -coated vesicles that emerge from transitional ER sites. The COPII coat, which consists of the small GTPase Sar1p, Sec23/24 complex, and Sec13/31 complex, selects vesicle cargo through recognition of export signals and forms ER-derived vesicles through assembly of an outer layer cage structure (1.Lee M.C. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (726) Google Scholar, 2.Gürkan C. Stagg S.M. Lapointe P. Balch W.E. Nat. Rev. Mol. Cell Biol. 2006; 7: 727-738Crossref PubMed Scopus (176) Google Scholar). Cytoplasmically exposed ER export signals have been identified in secretory cargo including the C-terminal dihydrophic and diacidic motifs (3.Kappeler F. Klopfenstein D.R. Foguet M. Paccaud J.P. Hauri H.P. J. Biol. Chem. 1997; 272: 31801-31808Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 4.Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (402) Google Scholar). Structural studies indicate that the Sec24p subunit of the COPII coat contains distinct binding sites for some of the molecularly defined export signals (5.Mossessova E. Bickford L.C. Goldberg J. Cell. 2003; 114: 483-495Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 6.Miller E.A. Beilharz T.H. Malkus P.N. Lee M.C. Hamamoto S. Orci L. Schekman R. Cell. 2003; 114: 497-509Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Thus a cycle of cargo-coat interactions regulated by the Sar1p GTPase directs anterograde movement of secretory proteins into ER-derived transport vesicles (7.Sato K. Nakano A. FEBS Lett. 2007; 581: 2076-2082Crossref PubMed Scopus (167) Google Scholar). coat protein complex II alkaline phosphatase coat protein complex I endoplasmic reticulum wild type hemagglutinin dithiobissuccinimidyl propionate soluble N-ethylmaleimide-sensitive factor attachment protein receptor. Although many secretory proteins contain known export signals that interact directly with COPII subunits, the diverse array of secretory cargo that depends on this export route requires additional machinery for efficient collection of all cargo into COPII vesicles (1.Lee M.C. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (726) Google Scholar). For instance certain soluble secretory proteins as well as transmembrane cargo require protein sorting adaptors for efficient ER export. These membrane-spanning adaptors, or sorting receptors, interact directly with secretory cargo and with coat subunits to efficiently couple cargo to the COPII budding machinery. For example, ERGIC-53 acts as a protein sorting adaptor for several glycoproteins and has a large N-terminal lumenal domain that interacts with secretory proteins including blood coagulation factors, cathepsins, and α1-antitrypsin (8.Nichols W.C. Seligsohn U. Zivelin A. Terry V.H. Hertel C.E. Wheatley M.A. Moussalli M.J. Hauri H.P. Ciavarella N. Kaufman R.J. Ginsburg D. Cell. 1998; 93: 61-70Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 9.Appenzeller C. Andersson H. Kappeler F. Hauri H.P. Nat. Cell Biol. 1999; 1: 330-334Crossref PubMed Scopus (282) Google Scholar, 10.Nyfeler B. Reiterer V. Wendeler M.W. Stefan E. Zhang B. Michnick S.W. Hauri H.P. J. Cell Biol. 2008; 180: 705-712Crossref PubMed Scopus (102) Google Scholar). The cytoplasmic C-terminal tail of ERGIC-53 contains a diphenylalanine export signal that is necessary for COPII export as well as a dilysine motif required for COPI-dependent retrieval to the ER (11.Hauri H.P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Crossref PubMed Google Scholar). Additional ER vesicle proteins identified in yeast have been shown to interact with the COPII coat as well as specific secretory proteins (12.Otte S. Belden W.J. Heidtman M. Liu J. Jensen O.N. Barlowe C. J. Cell Biol. 2001; 152: 503-518Crossref PubMed Scopus (135) Google Scholar). For example Erv29p acts as a protein sorting adaptor for the soluble secretory proteins glyco-pro-α-factor and carboxypeptidase Y (13.Belden W.J. Barlowe C. Science. 2001; 294: 1528-1531Crossref PubMed Scopus (164) Google Scholar). Erv29p also contains COPII and COPI sorting signals that shuttle the protein between ER and Golgi compartments. More recently Erv26p was identified as a cargo receptor that escorts the pro-form of secretory alkaline phosphatase (ALP) into COPII-coated vesicles (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). Although COPII sorting receptors have been identified, the molecular mechanisms by which these receptors link cargo to coat remain poorly understood. Moreover it is not clear how cargo binding is regulated to promote interaction in the ER and then trigger dissociation in the Golgi complex. We have shown previously that Erv26p binds to pro-ALP and is required for efficient export of this secretory protein from the ER (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). Therefore specific lumenal regions of Erv26p are proposed to interact with pro-ALP, whereas cytosolically exposed sorting signals are presumably recognized and bound by coat subunits. To gain insight on the molecular contacts required for Erv26p sorting function, we undertook a systematic mutational analysis of this multispanning membrane protein. After generating a series of Erv26p mutants, we observed that mutation of specific residues in the third loop domain affect pro-ALP interaction and that residues in the C-terminal cytosolic tail are required for COPII and COPI transport. Finally mutation of residues in the second loop domain influenced Erv26p homodimer formation and sorting activity. Strains used in this study are listed in Table 1. Cells were grown in rich medium (1% Bacto yeast extract, 2% Bacto peptone, 2% dextrose) or minimal medium (0.67% yeast nitrogen base without amino acids, 2% dextrose) with appropriate supplements at 30 °C unless otherwise noted. Standard yeast (15.Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2598) Google Scholar) and bacteria (16.Ausubel R.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987: 3.0.1-3.14.3Google Scholar) molecular genetic methods were used.TABLE 1Yeast strains used in this studyStrainGenotypeSource or Ref.CBY740MATα his3 leu2 lys2 ura3Research Genetics BY4742CBY1480MATα his3 leu2 lys2 ura3 erv26ΔKANRResearch Genetics 16420CBY1912CBY1480 with pRS316-ERV2614.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google ScholarCBY1471Matα his3 leu2 lys2 ura3 HIS3MX6-Gal1-3XHA-ERV26This studyCBY2150MATα his3 leu2 lys2 ura3 Erv26-3HA-HIS3MX6This studyCBY2076MATα his3 leu2 lys2 ura3 PHO8-3XHA-HIS3MX614.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google ScholarCBY2305CBY2150 with pRS316-ERV26This studyCBY2462MATα his3 leu2 lys2 ura3 PHO8-3XHA-HIS3MX6 erv26ΔKANR with pRS316-ERV26This studyCBY2891CBY1480 with pRS316-ERV26-LLEL2-5AAAAThis studyCBY2617CBY1480 with pRS316-ERV26-LL2-3AAThis studyCBY2616CBY1480 with pRS316-ERV26-L3EThis studyCBY2366CBY1480 with pRS316-ERV26-E4AThis studyCBY2618CBY1480 with pRS316-ERV26-LL5-6AAThis studyCBY2367CBY1480 with pRS316-ERV26-E32AThis studyCBY2315CBY1480 with pRS316-ERV26-EE35-36AAThis studyCBY2368CBY1480 with pRS316-ERV26-RR42-43AAThis studyCBY2596CBY1480 with PRS316-ERV26-KL67-68ATThis studyCBY2595CBY1480 with pRS316-ERV26-K67NThis studyCBY2628CBY1480 with pRS316-ERV26-S72AThis studyCBY2597CBY1480 with pRS316-ERV26-I73NThis studyCBY2303CBY1480 with pRS316-ERV26-C75MThis studyCBY2342CBY1480 with pRS316-ERV26-FSI71-73AAAThis studyCBY2308CBY1480 with pRS316-ERV26-YIVYY76-80AAAAAThis studyCBY2629CBY1480 with pRS316-ERV26-Y76AThis studyCBY2460CBY1480 with pRS316-ERV26-YY79-80AAThis studyCBY2631CBY1480 with pRS316-ERV26-L83NThis studyCBY2598CBY1480 with pRS316-ERV26-Q81AThis studyCBY2640CBY1480 with pRS316-ERV26-N82GThis studyCBY2599CBY1480 with pRS316-ERV26-K84AThis studyCBY2892CBY1480 with pRS316-ERV26-NH106-107AAThis studyCBY2369CBY1480 with pRS316-ERV26-H107AThis studyCBY2317CBY1480 with pRS316-ERV26-YF114-115AAThis studyCBY2365CBY1480 with pRS316-ERV26-K112AThis studyCBY2600CBY1480 with pRS316-ERV26-F114AThis studyCBY2601CBY1480 with pRS316-ERV26-D116AThis studyCBY2602CBY1480 with pRS316-ERV26-E118AThis studyCBY2316CBY1480 with pRS316-ERV26-P120AThis studyCBY2603CBY1480 with PRS316-ERV26-Q122AThis studyCBY2604CBY1480 with pRS316-ERV26-K124AThis studyCBY1996CBY1480 with pRS316-ERV26-E218AThis studyCBY1997CBY1480 with pRS316-ERV26-E218A-E220AThis studyCBY1995CBY1480 with pRS316-ERV26-D222AThis studyCBY1994CBY1480 with pRS316-ERV26-D222A-D224AThis studyCBY2893CBY1480 with pRS316-ERV26-RKYIYSL205-211AAAAAAAThis studyCBY2894CBY1480 with pRS316-ERV26-RVVINSV198-204AAAAAAAThis studyCBY2895CBY1480 with pRS136-ERV26-VV199-200TTThis studyCBY2896CBY1480 with pRS316-ERV26-V228DThis studyCBY2897CBY1480 with pRS316-ERV26-ΔRLAV (residues 225–228)This studyCBY2047MATα his3 leu2 lys2 ura3 Erv26ΔC1(Δ173–228)-3HA-HIS3MX6This studyCBY2847MATα his3 leu2 lys2 ura3 Erv26ΔC2(Δ162–228)-3HA-HIS3MX6This studyCBY2548MATα his3 leu2 lys2 ura3 KTR3-3XHA-HIS3MX6This studyCBY2767MATα his3 leu2 lys2 ura3 KTR3-3XHA-HIS3MX6 erv26ΔKANR with pRS316-ERV26-P120AThis studyCBY2768MATα his3 leu2 lys2 ura3 KTR3-3XHA-HIS3MX6 erv26ΔKANR with pRS316-ERV26-Q122AThis studyCBY2322MATα his3 leu2 lys2 ura3 PHO8-3XHA-HIS3MX6 erv26ΔKANR with pRS316-ERV26-P120AThis studyCBY2622MATα his3 leu2 lys2 ura3 PHO8-3XHA-HIS3MX6 erv26ΔKANR with pRS316-ERV26-Q122AThis studyCBY2319CBY2150 with pRS316-ERV26-P120AThis studyCBY2755MATα his3 leu2 lys2 ura3 PHO8-3XHA-HIS3MX6 erv26ΔKANR with pRS316-ERV26-I73NThis studyCBY2758MATα his3 leu2 lys2 ura3 PHO8-3XHA-HIS3MX6 erv26ΔKANR with pRS316-ERV26-C75MThis studyCBY2762CBY2150 with pRS316-ERV26-I73NThis studyCBY2304CBY2150 with pRS316-ERV26-C75MThis study Open table in a new tab Construction of pRS316-ERV26 was described previously (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). Mutagenesis of pRS316-ERV26 was performed using the QuikChange site-directed mutagenesis method (Stratagene), and mutants were verified by sequencing. Oligonucleotide primers used in this study are available upon request. The erv26Δ::KAN and ALP-HA strains have been described previously (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). Ktr3-HA was constructed by targeting the KTR3 gene with the PCR product generated from pFA6a-3HA-His3MX6 (17.Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4281) Google Scholar) using primers Ktr3-HA.F2 and Ktr3-HA.R1. Erv26-HA was constructed using the same method targeting the ERV26 gene with the PCR product of pFA6a-3HA-His3MX6 using primers Erv26-HA.F2 and Erv26-HA.R1. HA-Erv26 was constructed by targeting the ERV26 gene with the PCR product of pFA6a-His3MX6-PGAL1–3HA using primers YHR181W-F4 and YHR181W-R3. Antibodies directed against Erv25p (18.Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), Sec61p (19.Stirling C.J. Rothblatt J. Hosobuchi M. Deshaies R. Schekman R. Mol. Biol. Cell. 1992; 3: 129-142Crossref PubMed Scopus (280) Google Scholar), Kar2p, Och1p, Erv41p, Erv46p (12.Otte S. Belden W.J. Heidtman M. Liu J. Jensen O.N. Barlowe C. J. Cell Biol. 2001; 152: 503-518Crossref PubMed Scopus (135) Google Scholar), Sec22p (20.Liu Y. Barlowe C. Mol. Biol. Cell. 2002; 13: 3314-3324Crossref PubMed Scopus (113) Google Scholar), ALP (21.Haas A. Scheglmann D. Lazar T. Gallwitz D. Wickner W. EMBO J. 1995; 14: 5258-5270Crossref PubMed Scopus (235) Google Scholar), and Erv26p (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar) have been described previously. Immunoblots were developed using SuperSignal West Pico detection reagents (Thermo Scientific) and developed with a UVP bioimaging system (Upland, CA). Yeast semi-intact cell membranes (22.Baker D. Hicke L. Rexach M. Schleyer M. Schekman R. Cell. 1988; 54: 335-344Abstract Full Text PDF PubMed Scopus (259) Google Scholar) and microsomal membranes (23.Wuestehube L.J. Schekman R.W. Methods Enzymol. 1992; 219: 124-136Crossref PubMed Scopus (80) Google Scholar) were isolated as described previously. Subcellular fractionation of organelles was performed as described previously (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). Microsomes (0.75 mg of membrane protein/ml final) in Buffer 88 (20 mm HEPES, pH 7.0, 250 mm sorbitol, 150 mm KOAc, 5 mm MgOAc) were mixed with or without proteinase K (final concentration, 0.2 mg/ml). Where noted, 0.2% Triton X-100 was included. Reactions were incubated for 40 min on ice and stopped by addition of 2 mm phenylmethylsulfonyl fluoride. One volume of 5× SDS-PAGE sample buffer was added, and proteins were resolved on 12.5% polyacrylamide gels for immunoblot analysis. Microsomes (0.85 mg of membrane protein/ml final) were mixed with Buffer 88 and increasing concentrations of dithiobissuccinimidyl propionate (DSP) in dimethyl sulfoxide (0.1–0.4 mm DSP) or DMSO alone and incubated for 20 min at 20 °C. Cross-linking was quenched by incubation with 2.5 mm Tris, 20 mm glycine on ice for 10 min. Reactions were resolved on polyacrylamide gels and analyzed by immunoblot. Pulse-chase experiments were performed as described previously (18.Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Briefly cells were grown in reduced sulfate media to an A600 of 0.4, harvested, washed and resuspended at an A600 of 3.0 in minimal media without sulfate. After preincubation at 30 °C for 5 min, cultures were pulsed with 35S-PRO-MIX (Amersham Biosciences) at 25 μCi/A600 cells. The chase phase was initiated after 7 min by the addition of excess unlabeled methionine and cysteine. ALP and carboxypeptidase Y were immunoprecipitated from common extracts and resolved on 8% polyacrylamide gels, and labeled proteins visualized by phosphorimaging (GE Healthcare). To estimate half-times, the percentage of labeled ALP that had been processed to the mature form was plotted over time. Immunoprecipitation experiments were performed as described previously (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar) with the following modifications. Wild-type microsomes or semi-intact cells (220 μg of total membrane protein in 225 μl) were solubilized in an equal volume of Buffer 88, pH 8.0 containing 2% digitonin (B88-8/2.0% digitonin) or buffer 88, pH 7.0 containing 2% Triton X-100 (B88/2.0% TX100) as indicated in the presence of 1 mm phenylmethylsulfonyl fluoride and 5 mm EDTA. Digitonin-containing samples were centrifuged at 14,000 rpm for 2 min at room temperature, and Triton X-100-containing samples were centrifuged at 60,000 rpm for 10 min at 4 °C to pellet unsolubilized material. The supernatant fluid (∼400 μl) was recovered, and 5% of this solubilized material was saved as a total sample. The remainder was diluted with 2 volumes of B88-8/0.5% digitonin or B88/0.2% TX100. To immunoprecipitate ALP-HA, Ktr3-HA, or Erv26-HA, 0.25 μg of the monoclonal HA.11 antibody (Sigma-Aldrich) and 40 μl of 20% protein A-Sepharose beads were added. To immunoprecipitate Erv26p, 0.25 μg of the polyclonal Erv26p antibody and 40 μl of 20% protein A-Sepharose beads were added. After binding for 70 min at 4 °C, immunocomplexes bound to Sepharose beads were washed four times with B88-8/0.5% digitonin or B88/0.2% TX100. Bound proteins were eluted from the beads by adding 20 μl of SDS-PAGE sample buffer followed by incubation at 75 °C for 3 min. Total and immunoprecipitated samples were resolved on polyacrylamide gels and analyzed by immunoblot. Recoveries were calculated based on densitometric analysis using the Image J program. Representative results are shown after multiple replicate experiments. Large scale in vitro budding was performed with microsomes in the absence or presence of purified COPII components (24.Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1078) Google Scholar). A 22-μl portion of total reactions and 220 μl of supernatant fractions containing vesicles were centrifuged at 60,000 rpm (TLA100.3 rotor, Beckman Instruments) to pellet membranes. Membrane pellets were resuspended in SDS-PAGE sample buffer, and equal volumes were loaded on 12.5% polyacrylamide gels for immunoblots. Where indicated, preimmune or anti-Erv26p antibodies were added to membranes and incubated on ice for 15 min prior to the addition of budding factors. Packaging efficiencies were calculated as a percentage of totals by densitometry. Erv26p is a non-essential transmembrane protein that is predicted to contain four transmembrane segments (25.Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9576) Google Scholar, 26.von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1424) Google Scholar) with cytosolic N and C termini. To test this arrangement, location of the N and C termini were examined. We had previously performed in vitro budding experiments in the presence of a polyclonal Erv26p antiserum, which was raised against the C-terminal 69 amino acid residues of Erv26p, and observed that these antibodies specifically inhibited uptake of Erv26p into COPII vesicles (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). This result suggested that the C-terminal tail of Erv26p faces the cytosol. To investigate topology, two epitope-tagged versions of Erv26p were generated, placing 3×HA tags on either the N or C terminus at the ERV26 locus (17.Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4281) Google Scholar). The tagged proteins were stably expressed and supported pro-ALP processing to near wild-type levels (Fig. 4 and data not shown). Microsomal membranes were prepared from both HA-Erv26 and Erv26-HA strains and then used in protease protection assays to examine the accessibility of epitope-tagged Erv26p to proteinase K in the absence and presence of detergent. As seen in Fig. 1A, proteinase K treatment of HA-Erv26p in the absence of detergent (lane 3) caused a marked reduction in HA-tagged protein but also produced a partially protected species that was shifted in size by ∼8 kDa when immunoblots were developed with anti-HA. This proteolytic product was not detected with the C terminus-specific Erv26p polyclonal antiserum and the size shift corresponds to the approximate size of the 69-amino acid C terminus. In the presence of detergent, HA-Erv26p was completely digested, and no partial digestion products were detected with either antibody (lane 4). These results indicate that the N-terminal HA tag was largely sensitive to protease treatment in intact microsomal membranes; however, a minor protease-protected fragment was also generated from HA-Erv26. Proteinase K treatment of C-terminally tagged Erv26-HA membranes (Fig. 1A, lane 7) digested virtually all Erv26-HA protein as detected in both anti-HA and anti-Erv26p immunoblots. Residual Erv26-HA was completely digested in the presence of detergent (lane 8). These observations indicate that the C-terminal HA tag was also sensitive to proteinase K and therefore was cytosolically exposed, consistent with previous evidence for antibody accessibility to the C terminus of Erv26p (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). As controls for membrane integrity and proteinase K activity in these experiments, Erv46p, a transmembrane protein with relatively short cytosolic segments and a large protected lumenal domain (27.Otte S. Barlowe C. EMBO J. 2002; 21: 6095-6104Crossref PubMed Scopus (80) Google Scholar), as well as the cytosol-facing SNARE protein Bos1p (28.Newman A.P. Groesch M.E. Ferro-Novick S. EMBO J. 1992; 11: 3609-3617Crossref PubMed Scopus (64) Google Scholar) were monitored. Upon protease treatment, Erv46p shifted to a protease-protected species of the expected size (27.Otte S. Barlowe C. EMBO J. 2002; 21: 6095-6104Crossref PubMed Scopus (80) Google Scholar), whereas Bos1p was fully digested (lanes 3 and 7). Addition of proteinase K and detergent caused digestion of all proteins examined (lanes 4 and 8). Collectively these observations indicate that the N and C termini of Erv26p are cytosolically exposed, consistent with the proposed topology model (Fig. 1B). Generation of a partially protected HA-Erv26 fragment in lane 3 likely arises through increased protease sensitivity of the C-terminal tail segment relative to the N terminus. In previous studies, we demonstrated that Erv26p was non-essential but required for efficient packaging of the vacuolar directed pro-ALP into ER-derived vesicles. To further investigate the mechanism by which Erv26p connects pro-ALP with the COPII coat, we systematically mutated specific amino acids in Erv26p, first targeting conserved residues outside of the transmembrane segments and then focusing on specific regions of the protein. Mutations were introduced into an ERV26-CEN plasmid, expressed in the erv26Δ background, and initially screened by immunoblot to determine Erv26p expression level and pro-ALP accumulation relative to wild type. Fig. 2 shows representative immunoblots of Erv26p stability and pro-ALP levels from a subset of mutations in the second and third loop domains. The complete results of the mutant analysis are provided in Table 2. Certain mutants, such as L3E and F71A/S72A/I73A severely destabilized Erv26p, but several other mutants, such as C75M and P120A, showed expression levels similar to wild-type Erv26p levels. Some of the stably expressed mutants also showed varying degrees of pro-ALP accumulation such as C75M (Fig. 2B) and F114A, D116A, P120A, and Q122A (Fig. 2A). To assess the kinetics of pro-ALP biogenesis, a subset of the pro-ALP-accumulating strains were further analyzed in pulse-chase experiments. Cells were pulsed with 35S-amino acids for 7 min to label newly synthesized proteins and chased with unlabeled amino acids to monitor ALP and carboxypeptidase Y maturation rates. As described previously (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar), ALP maturation displayed a severe delay in erv26Δ strains, whereas carboxypeptidase Y matured at normal rates (Fig. 2C). The P120A and Q122A mutants displayed moderate delays in ALP maturation with half-times of ∼10 and 14 min, respectively, compared with a wild-type rate of ∼6 min. In addition, the C75M mutation decreased pro-ALP maturation (Fig. 2D) to a half-time of ∼24 min. These reductions in pro-ALP processing indicated impaired Erv26p function; therefore we next examined specific mutants in cell-free assays to gain insight on the defects associated with single amino acid changes on Erv26p function.TABLE 2Analysis of Erv26p mutantsMutantALP accumulationaALP key: ++, pro-ALP accumulation approximately half the difference between ERV26 and erv26Δ; +, modest but obvious accumulation.Erv26p stabilitybErv26p key: −, modest destabilization (75–90% of wt); −−, significant destabilization (less than 75%).N-terminal tailL2A/L3A/E4A/L5A26Δ26ΔL2A/L3Awt−L3E++−−E4Awt−L5A/I6A+−First loop domainE32AwtwtE35A/E36AwtwtR42A/R43AwtwtSecond loop domainK67A/L68T+wtK67N+−−L70N+−F71A/S72A/I73A++−−S72AwtwtI73N+−C75M+−Y76A/I77A/V78A/Y79A/Y80A26Δ−−Y76Awt/+wtY79A/Y80A+−Q81AwtwtN82GwtwtL83Nwt−−K84AwtwtThird loop domainN106A/H107A26Δ26ΔH107NwtwtK112AwtwtY113A/F114A++−−F114A+wtD116A+wtE118AwtwtP120A+wtQ122A+wtK124A+wtC-terminal tailE218AwtwtE218A/E220Awt−D222AwtwtD222A/D224A++−−aacaa, amino acids. 205–211 to Alawtwtaa 198–204 to AlawtwtV199T/V200Twtwt at 24 °CV228Dwtwt at 24 °CΔRLAV (aa 225–228)wtwt at 24 °CErv26ΔC1-HA++wtErv26ΔC2-HA++wtErv26-HA+wta ALP key: ++, pro-ALP accumulation approximately half the difference between ERV26 and erv26Δ; +, modest but obvious accumulation.b Erv26p key: −, modest destabilization (75–90% of wt); −−, significant destabilization (less than 75%).c aa, amino acids. Open table in a new tab Recent studies have indicated that the lumenal region of pro-ALP as well as its proximity to the inner leaflet of the ER membrane is required for Erv26p-dependent ER export (49.Dancourt J. Barlowe C. Traffic. 2009; 10: 1006-1018Crossref PubMed Scopus (15) Google Scholar). To test whether amino acid residues in the third loop region of Erv26p are required for lumenal interactions between Erv26p and pro-ALP, we performed immunoprecipitation experiments with the P120A and Q122A mutants. Previous experiments demonstrated that wild-type Erv26p co-immunoprecipitated specifically with pro-ALP-HA (14.Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (31) Google Scholar). To assess co-immunoprecipitation of pro-ALP-HA with Erv26p mutants, semi-intact cells from pro-ALP-HA strains expressing wild-type Erv26p, Erv26-P120A, or Erv26-Q122A were solubilized in digitonin and immunoprecipitated with polyclonal Erv26p antiserum. In Fig. 3A, wild-type Erv26p co-immunoprecipitated pro-ALP-HA (2.2% of total); however, significantly less pro-ALP-HA was recovered with the P120A (0.08%) and Q122A (0.04%) mutants compared with wild type. Although more pro-ALP-HA was present in the total P120A and Q122A membrane extracts, association with these Erv26p mutants was clearly diminished. Erv41p, an ER/Golgi membrane protein, served as a negative control and confirmed specificity of the Erv26p association with pro-ALP-HA. The significant reduction in pro-ALP-HA co-immunoprecipitated with the P120A and Q122A loop domain mutants indicated that this region of Erv26p is important for cargo interactions. To assess whether mutations in the third loop domain affected other properties of Erv26p, we monitored packaging in COPII budding assays. Proteins that cycle between the ER and Golgi are packaged into COPII vesicles even in the absence of newly synthesized cargo (29.Yeung T. Barlowe C. Schekman R. J. Biol. Chem. 1995; 270: 30567-30570Abstract Full Text
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