ER Degradation of a Misfolded Luminal Protein by the Cytosolic Ubiquitin-Proteasome Pathway

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The complementing plasmid, containing a 3.95-kb insert, was isolated by transforming the der2-1 mutant strain (31) with a genomic library contained in plasmid yCPlac111 [Cvrckova F., Nasmyth K., EMBO J. 12, 5277 (1993).] and screening for complementing colonies as described (7). Sequencing this insert from both ends and subsequent search in the EMBL database revealed the presence of the genes MSS1, UBC7 (QRI8), and MAC1. Only the 1.1-kb Eco RI-Pst I subclone containing UBC7 (10, 11) was able to complement the der2-1 mutation. The presence of UBC7 was verified by sequencing. A cross of the der2-1 mutant with a ubc7 deletion strain (W303-CQ) (31) did not lead to complementation of the mutant phenotype in the resulting diploid. Sporulation of this diploid produced four spores, all of which exhibited the mutant phenotype, indicating that DER2 is identical to UBC7 (QRI8) (8).

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For pulse-chase experiments, cells corresponding to 2 units of optical density at 600 nm (OD600) (0.6 × 108 to 0.8 × 108 cells) were taken from a logarithmically growing culture for each time point and labeled with [³⁵S]methionine. Growth, labeling, and chase conditions, as well as all other experimental procedures (cell breakage, immunoprecipitation, and SDS-PAGE), were as described (6).

The wild-type strains used in this study were W303-1B (MATα ade2-1oc ura3-1 his3-11,15 leu2-3,112 trp1-1 can1-100) [Chiang H. L., Schekman R., Nature 350, 313 (1991).], WCG4a (MATα his3-11,15 leu2-3,112 ura3) (21), and YPH499 (MATa ura3-52 leu2Δ1 his3Δ200 trp1Δ63 lys2-801 ade2-101) (22). Strain der2-1 was derived from ethylmethane sulfonate mutagenesis of cells of strain YAF6 (MATa pra1ΔSS prc1-1 leu2-3, 112 URA3 HIS3). Strains WCGY4a and YPH499Y were derived from strains WCG4a and YPH499 by replacing the chromosomal PRC1 allele with the prc1-1 allele (encoding CPY*) by the two-step gene replacement method [Scherer S., Davis R. W., Proc. Natl. Acad. Sci. U.S.A. 76, 4951 (1979).] with Bgl II-digested DNA of plasmid pRS306prc1-1 (7). The mutant strains CMY762Y (MATa cim3-1 ura3-52 leu2Δ1 his3Δ200 prc1-1), CMY806Y (MATa cim5-1 ura3-52 leu2Δ1 his3Δ200 prc1-1), WCGY4-11a (MATα his3-11,15 leu2-3,112 ura3 pre1-1 prc1-1), WCGY4-11/22a (MATα his3-11,15 leu2-3,112 ura3 pre1-1 pre2-2 prc1-1), and YHIY29/14 (MATα his3-11,15 leu2-3,112 ura3 pre1-1 pre4-1 prc1-1) were derived from the respective mutants CMY762, CMY806 (22), WCG4-11a, WCG4-11/22a (21), and YHI29/14 (W. Hilt and D. H. Wolf, unpublished data) by replacing the PRC1 wild-type allele with the prc1-1 allele, as described above. Strains W303-1C (MATα ade2-1^oc ura3-1 his3-11,15 leu2-3,112 trp1-1 can1-100 prc1-1) and W303-CD, derived from W303-1C but harboring the der1::URA3 mutation, were provided by M. Knop (7). Strain CB018 (MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1^oc can1-100 pep4Δ::HIS3 prb1::hisG prc1Δ::hisG) was provided by S. Fuller. The ubc6 deletion strain W303-CP was constructed by transforming cells of strain W303-1C with Hind III-digested DNA of plasmid pTX33 (ubc6::LEU2) (13) followed by selection for Leu⁺ transformants. The ubc7 deletion strain W303-CQ was constructed by transforming cells with Pst I- and Bam HI-digested DNA of plasmid pGR172 (ubc7::LEU2) (10) followed by selection for Leu⁺ transformants. In all instances, the homologous recombination events were confirmed by Southern (DNA) blot analysis. W303-CQa was derived from W303-CQ through mating type switching, with the use of the plasmid pGalHO [Hicks J. B., Herskowitz I., Genetics 83, 245 (1976).], and subsequent screening for the a-mating type. W303-CPQ and W303-CDQ were derived from crosses of W303-CQa with W303-CP and W303-CD, respectively. For all strain constructions, standard yeast genetic techniques were used [Guthrie C., Fink G. R., Eds., Methods Enzymol. 194 (1991)]. The 1.1-kb Eco RI-Pst I genomic fragment containing the UBC7 gene, made blunt at the Pst I end, was subcloned into the Eco RI- and Eco RV-digested CEN plasmid pRS313 [Sikorski R. S., Hieter P., Genetics 122, 19 (1989).] or 2μ plasmid pRS423 [Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P., Gene 110, 119 (1992).] to yield the plasmids pRS313-UBC7 and pRS423-UBC7, respectively. The plasmids Yep96, Yep110, and Yep112 contain WT-Ub, Ub-R48 and HA-Ub under the control of the CUP1 promoter on a 2μ plasmid (17). The plasmids containing Ub-R29 and Ub-R63 are derivatives of Yep96 (16).

Cells expressing the different ubiquitin variants from plasmids (31) were grown at 30°C in complete synthetic medium (CM) into log phase until an OD₆₀₀ of 2 units was achieved. They were then transferred into CM containing 0.1% glucose and 100 μM copper sulfate (OD₆₀₀, 10) and grown for 4 hours to allow for induction of CPY* and the synthesis of the respective ubiquitin variant. Cells (OD₆₀₀, 100 units) (3 × 10⁹ to 4 × 10⁹ cells) were subjected to alkaline lysis (0.25 M NaOH, 1% mercaptoethanol) for 10 min on ice, followed by precipitation with 10% (w/v) trichloroacetic acid (TCA) for 10 min on ice. After centrifugation (10 min, 20,000g), the resulting pellet was washed once with ice-cold ethanol, resuspended in 500 μl of UREA buffer [5% SDS, 8 M urea, 200 mM tris-HCl (pH 6.8), 0.1 mM EDTA, bromophenol blue], diluted 1 to 20, and subjected to immunoprecipitation with antibodies to CPY essentially as described previously (6). Deglycosylation of samples was performed as described [Spormann D. O., Heim J., Wolf D. H., J. Biol. Chem. 267, 8021 (1992).]. Immunoprecipitated material was boiled in 60 μl of UREA buffer before SDS-PAGE. CPY* antigenic material corresponding to 15 OD₆₀₀ units of cells (4.5 × 10⁸ cells) was separated on an 8% SDS-polyacrylamide gel and transferred to nitrocellulose membranes according to standard methods. HA-Ub was detected with the 12CA5 antibody to HA and enhanced chemiluminescence (ECL; Amersham). Kar2p was similarly detected with specific polyclonal antibodies.

Cells were grown as described (32). Spheroplast generation and cell breakage were performed essentially as described [Egner R., Mahe Y., Pandjaitan R., Kuchler K., Mol. Cell. Biol. 15, 5879 (1995).] with an additional treatment with 20 mM N-ethylmaleimide before spheroplast generation. Spheroplasts were generated from 300 OD₆₀₀ units of cells and gently lysed with a tissue grinder. The lysate was cleared of remaining cells and debris by repeated centrifugation for 5 min at 3000g, and the final supernatant was divided into portions corresponding to 60 OD₆₀₀ units of cells. From the time of lysis, all material was maintained on ice. Membranes were separated from the soluble fraction by centrifugation at 20,000g for 30 min at 4°C. For protease treatment, the pellet was resuspended and trypsin was added to a concentration of 0.5 mg/ml. The sample was incubated for 30 min on ice (if added, Triton X-100 was present at 1% during this incubation). All treatments were terminated by precipitation of proteins with 10% TCA for 10 min on ice. Solubilization of the TCA pellet, immunoprecipitation of CPY* antigenic material, SDS-PAGE, and detection of HA-Ub-conjugated CPY* were performed as described (32).

The separation of unglycosylated from glycosylated proteins by Con A-Sepharose (Sigma) was performed in Con A buffer [0.01 M tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM CaCl2, 1 mM MnCl2]. Immunoprecipitated CPY* antigenic material was solubilized in UREA buffer (32), diluted 1:20 in Con A buffer, and incubated with Con A-Sepharose (10 to 16 mg of Con A per milliliter of gel; 1.1 μl of gel per OD₆₀₀ unit of cells) for 3 hours at room temperature with gentle agitation. The Con A-Sepharose was separated by centrifugation (1500g, 3 min) and washed twice with Con A buffer. Proteins of the combined supernatant and washes were precipitated with 10% TCA for 10 min on ice and solubilized in UREA buffer (32), as were the proteins bound to Con A-Sepharose. SDS-PAGE and detection of HA-Ub-conjugated CPY* were performed as described (32).

We thank H. L. Chiang, S. Fuller, W. Heinemeyer, W. Hilt, M. Knop, and C. Mann for providing strains; F. Cvrckova and K. Nasmyth for the CEN-LEU2 library; M. J. Ellison, G. Faye, M. Hochstrasser, and S. Jentsch for providing plasmids; and H. Rudolph and R. Schekman for the antibodies to HA and to Kar2p, respectively. Supported by the Bundesministerium für Forschung und Technologie (ZSP Stuttgart grant B 3.7 U) and the Fonds der Chemischen Industrie, Frankfurt.