Distinct Roles for the Cytoplasmic Tail Sequences of Emp24p and Erv25p in Transport between the Endoplasmic Reticulum and Golgi Complex
2001; Elsevier BV; Volume: 276; Issue: 46 Linguagem: Inglês
10.1074/jbc.m108113200
ISSN1083-351X
AutoresWilliam J. Belden, Charles Barlowe,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoHeteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K d ∼100 μm), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER. Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K d ∼100 μm), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER. coat protein complex II endoplasmic reticulum coat protein complex I polymerase chain reaction kilobase(s) splicing by overlay extension polyacrylamide gel electrophoresis N,N-dimethylformamide coupling buffer glycosylphosphatidylinositol The secretory pathway in eukaryotic cells consists of a series of membrane-bound compartments that modify, sort and transport secretory cargo. Transport through this pathway depends on coat protein complexes that form vesicles and select specific cargo molecules for incorporation into vesicles. Current models suggest that transport between organelles is bi-directional, such that organellar constituents are recycled as secretory cargo advances. With regard to transport through the early secretory pathway, coat protein complex II (COPII)1 catalyzes anterograde transport between the ER and Golgi whereas coat protein complex I (COPI) acts in retrograde traffic between these compartments (1Mellman I. Warren G. Cell. 1999; 100: 99-112Abstract Full Text Full Text PDF Scopus (361) Google Scholar). In addition to coat-dependent export of secretory cargo from the ER, retrieval (2Semenza J.C. Hartwick K.G. Dean N. Pelham H.R.B. Cell. 1990; 61: 1349-1357Abstract Full Text PDF PubMed Scopus (389) Google Scholar) and retention (3Sato M. Sato K. Nakano A. J. Cell Biol. 1996; 134: 279-294Crossref PubMed Scopus (128) Google Scholar) mechanisms operate to maintain overall compartmental organization. A related group of integral membrane proteins, referred to as the p24 family, are thought to act in concert with COPI and COPII to sort proteins during transport through the early secretory pathway. Initially identified on ER membranes (4Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar) and subsequently detected as abundant proteins on COPI- and COPII-coated vesicles (5Stamnes M.A. Craighead M.W. Hoe M.H. Lampen N. Geromanos S. Tempst P. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8011-8015Crossref PubMed Scopus (196) Google Scholar, 6Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (283) Google Scholar), the function of p24 proteins in sorting remains unclear. In yeast strains lacking certain p24 members, some secretory proteins accumulate in the ER (e.g. invertase and the GPI-anchored protein Gas1p), while ER resident proteins that contain an HDEL retrieval motif are secreted and the unfolded protein response pathway is activated (6Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (283) Google Scholar, 7Elrod-Erickson M.J. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1043-1058Crossref PubMed Scopus (143) Google Scholar, 8Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Belden W.J. Barlowe C. Mol. Biol. Cell. 2001; 12: 957-969Crossref PubMed Scopus (82) Google Scholar). Based on these and other findings, the p24 proteins have been proposed to act as structural components of vesicles (10Bremser M. Nickel W. Schweikert M. Ravazzola M. Amherdt M. Hughes C.A. Sollner T.H. Rothman J.E. Wieland F.T. Cell. 1999; 96: 495-506Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), as cargo receptors (11Muñiz M. Nuoffer C. Hauri H-P. Riezman H. J. Cell Biol. 2000; 148: 925-930Crossref PubMed Scopus (212) Google Scholar), as negative regulators of vesicle budding (7Elrod-Erickson M.J. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1043-1058Crossref PubMed Scopus (143) Google Scholar) or to establish specialized subdomains on organellar membranes (12Lavoie C. Paiement J. Dominguez M. Roy L. Dahan S. Gushue J.N. Bergeron J.J. J. Cell Biol. 1999; 146: 285-299Crossref PubMed Scopus (80) Google Scholar, 13Rojo M. Emery G. Marjomaki V. McDowall A.W. Parton R.G. Gruenberg J. J. Cell Sci. 2000; 113: 1043-1057Crossref PubMed Google Scholar). There are eight p24 proteins in yeast encoded by EMP24, ERV25, and ERP1-ERP6 (14Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J.M. Solari R.C.E. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (153) Google Scholar). Deletion of EMP24and/or ERV25 produce the strongest phenotypes with regard to the transport and sorting defects; however, deletion of theERP1 and ERP2 genes also display similar but weaker phenotypes. Indeed, evidence suggests that Emp24p, Erv25p, Erp1p, and Erp2p function in a heteromeric complex (8Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 14Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J.M. Solari R.C.E. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (153) Google Scholar). There are no apparent phenotypes associated with deletion of theERP3-ERP6 genes. Deletion of all eight p24-encoding genes in yeast produces viable cells with phenotypes that are indistinguishable from the single EMP24 or ERV25 deletions (15Springer S. Chen E. Duden R. Marzioch M. Rowley A. Hamamoto S. Merchant S. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4034-4039Crossref PubMed Scopus (95) Google Scholar). The p24 proteins are composed of a lumenally oriented amino-terminal domain and a single transmembrane segment that is followed by a cytoplasmically exposed ∼12-amino acid carboxyl-terminal sequence. Many of the carboxyl-terminal tail sequences found in p24 proteins possess dilysine motifs that are predicted to interact with subunits of the COPI coat and localize these proteins to the early secretory pathway. Indeed, binding assays using immobilized tail sequences from p24 family members indicate a role for dilysine motifs in COPI binding and that a double phenylalanine sequence present in many of these tail sequences facilitates binding to both COPI and COPII. Furthermore,in vivo evidence indicates a role for these motifs in proper localization of p24 proteins (16Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 271: 1396-1399Crossref Scopus (275) Google Scholar, 17Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (181) Google Scholar, 18Dominguez M. Dejgaard K. Fullerkrug J. Dahan S. Fazel A. Paccaud J-P. Thomas D.Y. Bergeron J.J.M. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (291) Google Scholar) although these analyses may be complicated by the affects of overexpression (13Rojo M. Emery G. Marjomaki V. McDowall A.W. Parton R.G. Gruenberg J. J. Cell Sci. 2000; 113: 1043-1057Crossref PubMed Google Scholar, 18Dominguez M. Dejgaard K. Fullerkrug J. Dahan S. Fazel A. Paccaud J-P. Thomas D.Y. Bergeron J.J.M. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (291) Google Scholar). It remains to be determined how distinct cytoplasmic tail sequences function in localizing p24 complexes and directing them into specific coated vesicles. In this report we focus on the cytoplasmic tail sequences contained on the Emp24p and Erv25p proteins to define the requirements for bi-directional transport of the Emp24p-Erv25p complex. A series of tail deletions and chimeras were generated and then monitored throughin vivo and in vitro assays. Further, the coat-binding properties of these isolated tail sequences were determined. Our results suggest that both the Emp24p and Erv25p tail sequences interact with the COPII coat and direct this complex into COPII vesicles, whereas the Erv25p tail sequence is required in COPI binding and retrograde transport from the Golgi complex to the ER. Yeast strains used in this study are listed in Table I and were grown in rich medium (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% dextrose) or selective medium (0.67% nitrogen base without amino acids, 2% dextrose, and required supplements). Other standard media and genetic methods used have been previously described (19Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). The Escherichia coli strain DH5α was used for manipulation of recombinant DNA and was grown in LB medium (1% NaCl, 1% Bacto-tryptone, and 0.5% Bacto-yeast extract) containing 100 μg/ml ampicillin if required.Table IStrains used in this studyStrainGenotypeReferenceFY833Mata his3Δ200 ura3–52 leu2Δ1 lys2Δ202 trp1Δ6321Winston F. Dollard C. Ricupero-Hovasse S.L. Yeast. 1995; 11: 53-55Crossref PubMed Scopus (784) Google ScholarFY834Matα his3Δ200 ura3–52 leu2Δ1 lys2Δ202 trp1Δ6321Winston F. Dollard C. Ricupero-Hovasse S.L. Yeast. 1995; 11: 53-55Crossref PubMed Scopus (784) Google ScholarRSY265Matα his4–619 ura3–52, sec13–123Kaiser C.A. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (545) Google ScholarBY4739Matα leu2Δ lys2Δ ura3Δ37Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. et al.Science. 1999; 285: 901-906Crossref PubMed Scopus (3191) Google ScholarBY4739-erp1BY4739 witherp1Δ∷KAN37Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. et al.Science. 1999; 285: 901-906Crossref PubMed Scopus (3191) Google ScholarCBY99FY834 withemp24Δ∷LEU28Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google ScholarCBY112FY834 withemp24Δ∷LEU2, erv25∷HIS38Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google ScholarCBY114FY834 with erv25Δ∷HIS38Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google ScholarCBY241CBY114 with pRV306 (ERV25 in pRS306)This studyCBY242CBY114 with pEVS306 (EVS in pRS306)This studyCBY243CBY114 with pEVE306 (EVE in pRS306)This studyCBY244CBY99 with pRMP304 (EMP24 in pRS304)This studyCBY245CBY99 with pEME304 (EME in pRS304)This studyCBY289CBY99 with pEMS304 (EMS in pRS304)This studyCBY294CBY112 with pEVE306 and pEME304This studyCBY325FY834 with sec13–1This studyCBY338CBY99 with sec13–1This studyCBY339CBY244 with sec13–1This studyCBY340CBY289 with sec13–1This studyCBY345CBY245 with sec13–1This studyCBY341CBY114 with sec13–1This studyCBY342CBY241 with sec13–1This studyCBY343CBY242 with sec13–1This studyCBY344CBY243 with sec13–1This studyCBY374CBY294 with sec13–1This study Open table in a new tab All of the genes and gene fusion constructs used in this study were generated by polymerase chain reaction (PCR) amplification of DNA unless otherwise stated. PCR-amplified DNA was purified, cleaved, and ligated into appropriate vectors according to manufactures specification. All constructs were sequenced to ensure that no errors were introduced during PCR amplification. The ERV25 gene was subcloned from pBEV2 (8Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) into the yeast integrating vector pRS306 (20Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) using the restriction endonucleasesPstI and EcoRI to produce pRV306. TheEMP24 gene was obtained by PCR amplification of chromosomal DNA isolated from Saccharomyces cerevisiae strain FY834 (21Winston F. Dollard C. Ricupero-Hovasse S.L. Yeast. 1995; 11: 53-55Crossref PubMed Scopus (784) Google Scholar) using oligonucleotides EM1 (5′-GGAATTCCTGAGAGATCGGGTCGC-3′) and EM2 (5′-CGGGATCCGTAAAAAGTATGAAACCG-3′), which correspond to nucleotides −121 to −103 and nucleotides 719 to 701, respectively. The oligonucleotides EM1 and EM2 were engineered to contain the restriction endonuclease recognition sites EcoRI and BamHI, respectively, which allowed for convenient insertion into the yeast shuttle vector pRS314 (20Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) producing pREM314. Additional 5′-DNA sequence was added to pREM314 by subcloning a 1.2-kilobase (kb)XhoI/SalI fragment from pSEY-BS22 (gift from H. Reizman) into the XhoI site, which is located in the multiple cloning region of pRS314, and the SalI site, which is located at nucleotide 394 in EMP24. An integrating version of this vector was constructed by transferring the 1.5-kbXhoI/BamHI fragment into pRS304 (20Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to produce pRMP304. The EME gene fusion was constructed using the geneSplicing by Overlap Extension (SOE) method (22Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene. 1989; 77: 61-68Crossref PubMed Scopus (2641) Google Scholar). The 5′-portion of the gene fusion, termed Eme-Awhich spans amino acids 1–193 of Emp24p, was PCR-amplified using pRMP304 as a template with an oligonucleotide corresponding to the T3 promoter and TS2 (5′-GTTCTTAAGGTAGTATATCTGGAAAAG-3′). The 3′ portion of the gene fusion, termed Eme-B which spans amino acids 202–211 of Erv25p, was PCR-amplified using pRV306 as a template with oligonucleotides TS1 (5′-TACTACCTTAAGAACTACTTCAAAACG-3′) and an oligonucleotide corresponding to the T7 promoter. The restriction endonuclease recognition site AlfII was engineered into the homologous portion of TS1 and TS2 at the fusion junction. The SOE method was performed by mixing PCR-amplified fragments Eme-Aand Eme-B with oligonucleotides T3 and T7 followed by another round of amplification. The resulting 1.5-kb gene fusion was gel-purified and subcloned into pRS304 using the restriction endonucleases XhoI and BamHI generating pEME304. The truncated EMP24 gene product, which lacks the 10-amino acid cytoplasmic tail, was made by first digesting the pEME304 vector with the restriction endonuclease AlfII. Linearized DNA was gel-purified, and the 3′-recessed ends were filled-in with Klenow Fragment (New England Biolabs) and ligated. The fill-in reaction created a stop codon at amino acid 194, and the vector was termed pEMS304. The EVE gene fusion was constructed using the SOE method. The 5′-portion of the gene fusion, termed Eve-A, that spans amino acids 1–201 of Erv25p was PCR-amplified using pRV306 as a template with oligonucleotides EV1 (5′-GGGAATTAGCGTACAAAGAGTTTCTG-3′) and TS4 (5′-TCTCCGGAGATAGTTGACTTGCCAAAC-3′). The 3′ portion of the gene fusion, termed Eme-B, which spans amino acids 194–203 of Emp24p, was PCR-amplified using pRMP304 as a template with oligonucleotides EM2 and TS3 (5′-AACTATCTCCGGAGATTCTTTGAGGTCACA-3′). The restriction endonuclease recognition site for BspEI was engineered into the homologous portion of TS3 and TS4 at the fusion junction. The SOE reaction was performed by mixing PCR-amplified fragmentsEme-A and Eme-B with oligonucleotides EV1 and EM2. The resulting 1.0-kb gene fusion was subcloned into pRS306 using the restriction endonucleases EcoRI and BamHI producing pEVE306. The EVS gene fusion also utilized the SOE method. The 5′-portion of the gene fusion termed EvsA was PCR-amplified using oligonucleotides EV1 and EVS2 (5′-TAGTTACTTAAGATAGTTGACTTGCCA-3′) from pBEV2. The 3′-portion of the gene fusion, termed EvsB,was also PCR-amplified from pBEV2 using oligonucleotide EVS1 (5′-TATCTTAAGTAACTACTTCAAAACG-3′) and the T7 oligonucleotide. The SOE reaction was performed by mixing equal concentrations ofEvsA and EvsB with the oligonucleotides EV1 and T7. The resulting 1.0-kb PCR-amplified DNA fragment was subcloned into pRS306 using the restriction endonucleases EcoRI andBamHI to produce pEVS306. CBY244 (Δ24-Emp24p), CBY245 (Δ24-EME), and CBY289 (Δ24-EMS) were made by transforming CBY99 (Δ24) with pRMP304, pEME304, or pEMS304 after linearization withSnaBI for targeting to the trp1Δ63 locus. CBY241 (Δ25-Erv25p), CBY243 (Δ25-EVE), and CBY242 (Δ25-EMS) were constructed by transforming CBY114 (Δ25) withStuI-linearized pRV306, pEVE306, or pEVS306 and targeted to the ura3–52 locus. CBY294 (Δ24/Δ25-EME,EVE) was made by first transforming CBY112 with linearized pEME304 and then transforming with linearized pEVE306. An isogenic set strains expressing the chimeric tail fusions and the sec13–1 temperature sensitive allele was generated by repeated backcrosses of RSY265 (23Kaiser C.A. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (545) Google Scholar). Sucrose gradient fractionation of membranes was performed as described with minor modifications (24Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (377) Google Scholar). Homogenized spheroplasts were centrifuged at 450 × gin Beckman SS34 rotor for 10 min, and 1 ml of the centrifuged cell lysate was layered on an 11-ml sucrose step gradient of 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, and 60 (weight/volume) sucrose in 10 mm Hepes, pH 7.0, and 1 mm MgCl2. The gradients were centrifuged at 45,000 rpm in a Beckman Ti45 rotor for 2.5 h, and then 13 fractions of 770 μl were collected. A 1:1 dilution of the fractions in 2× sample buffer were analyzed by SDS-PAGE, then transferred to nitrocellulose, and probed using antibodies against Sec61p (25Stirling C.J. Rothblatt J. Hosobuchi M. Deshaies R. Schekman R. Mol. Biol. Cell. 1992; 3: 129-142Crossref PubMed Scopus (280) Google Scholar), Emp47p (26Schroder S. Schimmoller F. Singer-Kruger B. Riezman H. J. Cell Biol. 1995; 131: 895-912Crossref PubMed Scopus (157) Google Scholar), Emp24p (6Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (283) Google Scholar), and Erv25p (8Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Reconstituted COPII budding reactions, measurement of Kar2p secretion, and intracellular accumulation of Gas1p were preformed as previously described by Belden and Barlowe (9Belden W.J. Barlowe C. Mol. Biol. Cell. 2001; 12: 957-969Crossref PubMed Scopus (82) Google Scholar). Synthetic peptides corresponding to the 11 carboxyl-terminal amino acids of Emp24p and Erv25p with an amino-terminal cysteine residue were generated (Biosynthesis Inc. Louisville, TX) and linked to thiopropyl-Sepharose 6B (Pharmacia) as described (17Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (181) Google Scholar) with minor modifications. Peptides with alanines substituted in Emp24p: FF-AA (CLRRAAEVTSLV), and SLV-AAA (CLRRFFEVTAAA): and Erv25p, YF-AA (CLKNAAKTKHII) were also generated. For the peptides corresponding to the Emp24p tails, the thiopropyl-Sepharose was swollen in 50% coupling buffer (CB) (0.1m Tris-HCl, pH 7.5, 0.5 m NaCl) with 50% DMF (CB-DMF) for 15 min at room temperature. The beads were then washed two times in CB-DMF and brought up to a final volume of 80% beads in CB-DMF. The bead solution (500 μl) was transferred to a Microfuge tube with 2 mg of peptide and incubated overnight at room temperature with constant mixing. Coupled peptides were washed two times with CB-DMF and once with blocking buffer (0.1 m Tris-HCL, pH 7.5, 0.5 m NaCl, 5.0 mm 2-mercaptoethonal, 50% DMF) and then incubated for 30 min at room temperature in blocking buffer. The beads were washed four times in CB-DMF and brought up to final volume of 80% beads in CB-DMF. The synthetic peptides corresponding to the Erv25p tail were handled in a similar fashion except DMF was excluded. Peptide coupling efficiency was monitored by measuring the absorbance at 343 nm according to the manufacturer's specifications. The amount of synthetic peptide bound to beads was quantified by a Lowery protein assay (27Ausubel F.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, New York1997: 3.0.1-3.14.3Google Scholar) using the synthetic peptides as standards. In vitro binding reactions were performed in a 0.5-ml Microfuge tube for 1 h at 4 °C with mixing. Equal amounts of individual COPII subunits, a COPII mixture, or a crude cytosol were incubated with equal amounts of peptide coupled to beads in 100 μl of reaction buffer (150 mm KOAC, 10 mm Hepes, pH 7.0, and 0.1% Triton X-100). After incubation, the beads were transferred to a new 1.5-ml Microfuge tube and washed six times with reaction buffer. After the final wash, the remaining buffer was removed with a Hamilton syringe, and the beads were resuspended in 15 μl of 2× sample buffer. Samples (7 μl) were resolved by SDS-PAGE, and bound proteins were detected by immunoblot. After quantifying bound protein by densitometry, equilibrium dissociation constants (K d) were calculated from double reciprocal plots of the data. Multiple cytoplasmic targeting sequences are present on p24 complexes but it is not known how distinct sequences function in transport. We sought to define the functional roles of two specific tail sequences from Emp24p and Erv25p in directing movement of this p24 complex between the ER and Golgi compartments. A set of Emp24p and Erv25p deletion and tail swap chimeras were constructed and expressed at endogenous levels. These constructs were analyzed for complementation in vivo by monitoring Gas1p accumulation, Kar2p secretion, and suppression of sec13–1 temperature sensitivity. To assess more precisely the consequences of these alterations, we determined the subcellular distribution of these proteins and used a cell-free budding assay to measure their packaging efficiency into COPII vesicles. Finally, the in vitrobinding properties of Emp24p and Erv25p tail peptides with COPI and COPII subunits were determined. An isogenic set of yeast strains expressing tail deletions and chimeras of the Emp24p-Erv25p complex were constructed as illustrated in Fig. 1. For example, the Δ24-EME strain (Emp24p with the Erv25p tail) expresses a chimeric EMP24 containing the COOH-tail of Erv25p in an emp24Δ strain. The Δ24-EMS strain (Emp24p with Stop codon at amino acid 194) expresses a truncated form of Emp24p lacking the final 10 amino acids at the carboxyl-terminal end in an emp24Δ strain. Both Δ24-EME and Δ24-EMS express an endogenous copy of ERV25. Similar approaches were used to generate the Δ25-EVE (Erv25p with Emp24 tail) and Δ25-EVS (Erv25p with stop codon) strains. Finally, the double swap strain (Δ/Δ-EME,EVE) carriesemp24Δ and erv25Δ alleles and expresses chimeric proteins from constructs integrated at TRP1 andURA3. Immunoblot analyses of membranes prepared from these strains were performed to assess the degree of complementation and the expression levels of the Emp24p and Erv25p proteins (Fig.2). Yeast strains lacking a functional Emp24p-Erv25p complex accumulate the GPI-anchored secretory protein Gas1p in the ER as a 105-kDa species. In wild-type strains, mature Gas1p migrates as a fully glycosylated 130-kDa protein (6Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (283) Google Scholar). As seen in the top panel of Fig. 2, no ER form of Gas1p was detected in the wild-type and Δ24-EME strains, whereas the other strains accumulated varying amounts of Gas1p in the ER. Densitometric scanning of these immunoblots indicated that ∼50% of the Gas1p contained in microsomes prepared from emp24Δ and erv25Δstrains migrated as the ER form. Similar levels were observed in the Δ25-EVE, Δ25-EVS, and Δ/Δ-EME,EVE strains. The Δ24-EMS strain accumulated an intermediate level of the ER form (∼35%) suggesting partial complementation. These results are summarized in TableII.Table IIPhenotypes of wild-type, p24 deletions, and chimerasWild-typeΔ24Δ24-EMEΔ24-EMSΔ25Δ25-EVEΔ25-EVSΔ/Δ-EME,EVEGas1p accumulation0%54%0%36%52%46%49%51%Kar2p secretion0%100%22%55%100%65%100%63%By-passsec13–1NOYESNOYESYESYESYESYESSubcellular localization (percent ER: percent Golgi) Sec61p74%:26%74%:26%80%:20%74%:26%88%:12%77%:23%76%:24%80%:20% Emp4732%:68%32%:68%28%:72%28%:72%27%:73%37%:63%26%:74%30%:70% Erv25p57%:43%63%:37%65%:35%60%:40%44%:56% Emp24p77%:23%45%:55%62%:38%Incorporation into COPII vesicles (percent of total packaged) Sec22p1213111113111112 Erv25p8.48.09.25.48.37.511 Emp24p8.88.84.84.05.26.54.5 Open table in a new tab We also monitored the expression levels of the Emp24p and Erv25p proteins. As observed previously (8Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 14Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J.M. Solari R.C.E. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (153) Google Scholar) their expression was interdependent such that emp24Δ and erv25Δreduced the levels of Erv25p and Emp24p, respectively. Near wild-type levels were observed in the Δ24-EME strain, and the other truncation or chimeric proteins were expressed at variably lower levels. Sec61p, an ER membrane protein that functions in polypeptide translocation, served as a loading control in these experiments to ensure equal analysis of samples. Extracellualr secretion of ER resident proteins (e.g. Kar2p) is another phenotype associated with emp24Δ anderv25Δ strains (7Elrod-Erickson M.J. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1043-1058Crossref PubMed Scopus (143) Google Scholar). Therefore, we quantified the amount of Kar2p secreted from these strains after a 3-h growth period, and a representative experiment is shown in Fig.3. Setting maximal Kar2p secretion levels at those observed for emp24Δ and erv25Δ, we found that the Δ25-EVS strain secreted near maximal levels, whereas Δ24-EMS, Δ25-EVE and Δ/Δ-EME,EVE secreted intermediate levels. The Δ24-EME strain secreted a low level of Kar2p that was near that of a wild-type strain. We next determined if our set of EMP24 and ERV25mutations could suppress the thermosensitivity of sec13–1strains. Previous studies showed that deletion of EMP24bypassed the requirement for SEC13 (7Elrod-Erickson M.J. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1043-1058Crossref PubMed Scopus (143) Google Scholar). Therefore, we crossed the sec13–1 mutation into our strains and scored for growth at 37 °C. As expected, both the emp24Δ anderv25Δ mutations suppressed sec13–1 (TableII). Interestingly, all of the mutations except Δ24-EME suppressedsec13–1 indicating that a partial loss of function mutation in EMP24 or ERV25 was adequate for suppression. All of these assays used to evaluate Emp24p/Erv25p function appear to show a close correlation; however, the Kar2p secretion assay seemed most sensitive followed by Gas1p accumulation then sec13–1suppression. Based on these assays, we conclude that the EME fusion protein (Emp24p with the Erv25p tail) functioned at near wild-type levels and that the EMS and EVE proteins displayed partial function, whereas the EVS protein did not provide detectable activity. A complete swap of the Emp24p and Er
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