Role of Cue1p in ubiquitination and degradation at the ER surface

Science

Volumn 278, Issue 5344, 1997, Pages 1806-1809

  Biederer, Thomas ^a   Volkwein, Corinna ^a   Sommer, Thomas ^a  

^a MAX DELBRÜCK CENTER FOR MOLECULAR MEDICINE   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

UBIQUITIN;

CELLULAR DISTRIBUTION; ENDOPLASMIC RETICULUM; IMMUNOFLUORESCENCE MICROSCOPY; MEMBRANE TRANSPORT; MICROSOME MEMBRANE; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN TRANSPORT; REVIEW;

AMINO ACID SEQUENCE; BIOLOGICAL TRANSPORT; CARBOXYPEPTIDASE C; CARBOXYPEPTIDASES; CARRIER PROTEINS; CYSTEINE ENDOPEPTIDASES; CYTOSOL; ENDOPLASMIC RETICULUM; INTRACELLULAR MEMBRANES; LIGASES; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; MULTIENZYME COMPLEXES; PROTEASOME ENDOPEPTIDASE COMPLEX; SACCHAROMYCES CEREVISIAE PROTEINS; UBIQUITIN-CONJUGATING ENZYMES; UBIQUITINS; YEASTS;

EID: 0030666729     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5344.1806     Document Type: Review

References (29)

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11. Preparation of ubiquitin-Sepharose and cytosolic fraction II, and performance of ubiquitin affinity chromatography, were carried out essentially as described [S. Jentsch, J. P. McGrath, A. Varshavsky, ibid. 329, 131 (1987)]. Ubiquitin-Sepharose was loaded with fraction II in the presence of 2.0 mM ATP; washed repeatedly with 25 mM tris (pH 7.4), 5 mM magnesium chloride, 2.0 mM ATP, and 0.2 mM DTT; and split into two halves. In parallel, microsomes were solubilized on ice with 25 mM tris (pH 7.5), 250 mM potassium acetate, 5 mM magnenesium chloride, 0.2 mM DTT, 2.0 mM ATP, and 1.0% CHAPS. One-half of the ubiquitin-Sepharose was loaded with the solubilized membrane proteins, the other with the solubilization buffer. After being washed with 25 mM tris (pH 7.5), 1.0 M potassium chloride, and 1.0% CHAPS, the columns were washed repeatedly with solubilization buffer. Elution of bound material was performed with solubilization buffer without ATP but containing 20 mM DTT. These eluates were further fractionated by RP-HPLC on an Aquapore 300 C8 column (Applied Biosystems) with an acetonitrile gradient. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
12. Microsome and cytosol preparations were performed essentially as described (4) [S. Panzner, L. Dreier, E. Hartmann, S. Kostka, T. A. Rapoport, Cell 81, 561 (1995)].
13. CUE1 was isolated from a genomic library (generated in the plasmid pSEY8) by hybridization with oligonucleotides. CUE1 is located on chromosome XIII (YMR264W). A null allele of CUE1 was constructed by amplification of 400 base pairs (bp) 5′ from the start codon and 650 bp 3′ from the stop codon by polymerase chain reaction (PCR) and insertion of the 2.2-kb fragment containing LEU2. A second disruption allele carries the HIS3 gene inserted into the singular Eco RI site of CUE1. Null alleles were introduced into yeast cells by the one-step gene disruption method and monitored by Southern (DNA) hybridization [F. M. Ausubel et al., Short Protocols in Molecular Biology (Greene Publishing Associates, New York, 1992)] Both disruptions showed no obvious growth defect (23).
14. 2-terminal cysteine. Affinity purification and immobilization of the antibodies were carried out as described (75). The antibodies detected a single protein of about 23 kD exclusively in crude microsomes of wild-type but not of Δcue1 cells (23). Protease protection assays of intact microsomes revealed that the COOH-terminus of Cue1p was oriented toward the cytosol (23).
15. Microsomes (10) were extracted as in (3).
16. GST-ubiquitin was expressed and immobilized as described [M. Scheffner, J. M. Huibregtse, R. D. Vierstra, P. M. Howley, Cell 75, 495 (1993)]. Binding of fraction II and CHAPS-solubilized membrane proteins was done on a small scale but essentially as in (9). The first elution was performed with 3.0 M urea and 1.0% CHAPS at room temperature, the second with nonreducing sample buffer containing 4.0% SDS. Bound GST-ubiquitin was not eluted with 3.0 M urea but with SDS (23). Binding of Cuep1 to GST-ubiquitin was dependent on the preloading step with fraction II (23). Fractions were analyzed by immunoblotting.
17. Immunoaffinity chromatography was performed essentially as described [D. Görlich, S. Prehn, E. Hartmann, K. U. Kalies, T. A. Rapoport, Cell 71, 489 (1992)]. Solubilization of microsomes and chromatography were performed in 25 mM tris (pH 7.5), 125 mM potassium acetate, 5 mM magnesium chloride, 1.0% Triton X-100 (Fluka, Buchs, Switzerland), 250 mM sucrose, 0.1 mM DTT, bovine serum albumin (1.0 mg/ml), 0.5 mM phenylmethytsulfonyl fluoride, leupeptin (10 μg/ml), and chymostatin (5 μg/ml).
18. myc, we also performed immunfluorescent microspcopy. A perinuclear ring-shaped staining was visible with antibodies to myc, which was indistinguishable from that of Kar2p (the ER-lumenal homolog of mammalian BiP). Cells that do not express the myc epitope showed virtually no staining (23).
19. myc, we also performed immunfluorescent microspcopy. A perinuclear ring-shaped staining was visible with antibodies to myc, which was indistinguishable from that of Kar2p (the ER-lumenal homolog of mammalian BiP). Cells that do not express the myc epitope showed virtually no staining (23).
20. Pulse-chase experiments and immunoprecipitation were done as described (4, 19). The wild-type strain used in this study was YWO2 (mata, trp1-1(am), his3-Δ200, ura3-52, lys2-801, leu2-3,-112). RSY521 (matα, leu2-3,-112, ura3-52, trp1-1, his4-401, HOL1-1) and YFP338 (matα, sec61-2, leu2-3,-112, ura3-52, ade2-3, pep4-3) were kindly supplied by M. Rose and R. Schekman. YTX5 (matα, Δubc6::HIS3, trp1-1(am), his3-Δ200, ura3-52, lys2-801, leu2-3,-112) and YTX93 (matα, sec61-2, Δubc7::LEU2, leu2-3,-112, ura3-52, ade2-3, pep4-3) were as described (4). Mutants used in this study [YTX105 (matα, Δcue1::HIS3, trp1-1(am), his3-Δ200, ura3-52, lys2-801, leu2-3,-112), YTX106 (matα, Δubc7::LEU2, trp1-1(am). his3-Δ200, ura3-52, lys2-801, leu2-3,-112), and YTX121 (matα, sec61-2, Δcue1::LEU2, leu2-3,-112, ura3-52, ade2-3, pep4-3)] were generated by direct transformation of null alleles (11). Null alleles of UBC6 and UBC7 have been described previously (3, 20). The prc1-1 allele was introduced into haploid wild-type cells as described (7) to generate YTX140 (mata, prc1-1, trp1-1(am), his3-Δ200, ura3-52, lys2-807, leu2-3,-112). Multiple mutants were generated by a second round of transformation [YTX141 (matα, Δcue1::HIS3, prc1-1, trp1-1(am), his3-Δ200, ura3-52, lys2-801, leu2-3,-112), YTX142 (mata, Δcue1::HIS3, Δubc7::LEU2, Δubc6::TRP1, prc1-1, trp1-1(am), his3-Δ200, ura3-52, lys2-801, leu2-3,-112)] or by crossing of single mutants and subsequent tetrad dissection [YTX133 (mata, Δcue1::HIS3, Δubc7::LEU2, trp1-1(am), his3-Δ200, ura3-52, lys2-801, leu2-3,-112)].
21. T. A. Rapoport, B. Jungnickel, U. Kutay, Annu. Rev. Biochem. 65, 271 (1996).
22. P. Chen, P. Johnson, T. Sommer, S. Jentsch, M. Hochstrasser, Cell 74, 357 (1993).
23. Growth of Δcue1 and Δcue1 cells overexpressing Ubc7p from a multicopy vector on minimal media containing cadmium (23) was tested as decribed in J. Jungmann, H. A. Reins, C. Schobert, S. Jentsch, Nature 361, 369 (1993).
24. Pulse-chase (4) and protease protection assays (3) were done essentially as described. The cells were lysed in 50 mM tris (pH 7.5), 250 mM sucrose, and 10 mM EDTA with one volume of glass beads by four repeated cycles of mixing with a Vortex for 30 s at maximum speed, interrupted by30-s incubations on ice. In every pulse-chase experiment, the extracts were untreated, treated with proteinase K (0.1 mg/ ml), or treated with proteinase K (0.1 mg/ml) and 0.4% Triton X-100 on ice for 15min. Noimmunoprecipitable CPY* was detected after treatment with proteinase K and detergent (23).
25. A. L. Schwartz, J. S. Trausch, A. Ciechanover, J. W. Slot, H. Geuze, Proc. Natl. Acad. Sci. U.S.A. 89, 5542 (1992); A. Palmer et al., Biochem. J. 316, 401 (1996).
26. A. L. Schwartz, J. S. Trausch, A. Ciechanover, J. W. Slot, H. Geuze, Proc. Natl. Acad. Sci. U.S.A. 89, 5542 (1992); A. Palmer et al., Biochem. J. 316, 401 (1996).
27. T. Biederer, C. Volkwein, T. Sommer, data not shown.
28. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L. Leu; M, Met; N, Asn; P. Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
29. We thank E. Hartmann for advice on the generation of Cue1p-specific antibodies; R. Kraft and S. Kostka for protein sequencing; M. Hochstrasser (Deg1-β-galactosidase fusion), S. Jentsch (Ubc1p antibodies, Δubc7::LEU, and pSEY8UBC7), T. A. Rapoport (Sec61p antibodies), M. Scheffner (GST-ubiquitin), and D. Wolf (prc1-1) for gene constructs and affinity-purified antibodies; and E, Hartmann, U. Kutay, A. Bergfeld, K. Breitschopf, T. A. Rapoport, and the members of the laboratory for helpful discussions and critical reading of this manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to T.S.