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A role for mammalian Ubc6 homologues in ER-associated protein degradation

Uwe Lenk¹, Helen Yu^2,*, Jan Walter¹, Marina S. Gelman², Enno Hartmann³, Ron R. Kopito² and Thomas Sommer^1,

¹ The Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13092 Berlin, Germany
² Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA
³ Universität Lübeck, Institut für Biologie, Ratzeburger Allee 160, 23538 Lübeck, Germany
^* Present address: Odyssey Pharmaceuticals Inc., 4550 Norris Canyon Road, Suite 140, San Ramon, CA 94583, USA

View larger version (33K): [in a new window]   Fig. 1. (A) The CLUSTALX-analysis of the indicated amino acid sequences demonstrates the two classes of Ubc6-related proteins. (B) Comparison of the amino acid sequences of scUbc6p, mmUbc6p and hsUbc6ep. The CLUSTALW-alignment displays the well-conserved N-terminal Ubc domain (marked by a dotted line) and the less conserved tail region in both Ubc6 subfamilies. The conserved active site cysteine is indicated by an asterisk and the region of the membrane spanning domains is underlined.

View larger version (45K): [in a new window]   Fig. 2. (A) Western blotting of membranes prepared from HEK 293 cells transiently transfected with a control plasmid or with expression vectors containing the indicated myc-tagged Ubc6 homologues. For unknown reasons the anti-myc antibody detected three bands for the epitope-tagged human homologue of the second class. (B) Immunofluorescence microscopy of HEK 293 cells expressing epitope-tagged versions of both classes of Ubc6 homologues. In the non-transfected control, anti-TRAP was used to stain a typical ER-membrane protein. The anti-myc antibody displays a very similar staining pattern for both Ubc6 classes.

View larger version (35K): [in a new window]   Fig. 3. (A) Epitope-tagged mmUbc6p and hsUbc6ep are integral membrane proteins, as is the control TRAP. Membrane preparations were extracted with buffer containing 2.5 M urea or 800 mM potassium acetate, with Na2CO3 (pH 11.6), or with 1.0% detergent. P, pellet; S, supernatant. (B) The ubiquitin-conjugating domain of both myc-tagged mmUbc6p and hsUbc6ep is orientated to the cytosol. Protease protection assay of myc-mmUbc6 and myc-hsUbc6p and the ER-lumenal control GRP78 (Santa Cruz Biotechnology, Santa Cruz, CA) and the integral ER membrane protein TRAP. Inside-out vesicles prepared from cells expressing the epitope-tagged Ubc6 homologues were treated with proteinase K or treated with 1% Triton X-100 and proteinase K for the indicated time. Samples were subjected to western blotting using the indicated antibodies.

View larger version (28K): [in a new window]   Fig. 4. (A) Wild-type and sec61-2 yeast strains were transformed with high-copy plasmids coding for Ubc6p or Ubc6pser or with an empty vector as indicated. Plates were incubated at 30 and 36.5°C as indicated. (B) A yeast strain coding for the mutant carboxypeptidase Y (CPY*) was transformed with high-copy plasmids coding for scUbc6p () or scUbc6pser () or with an empty () vector. Equal amounts of cells were labeled with 35S-cysteine/methionine and chased with non-radioactive medium. Aliquots were taken after 0, 30, 60 and 90 minutes chase-time and immunoprecipitated for CPY*, subjected to SDS-PAGE and analyzed by autoradiography. A typical experiment is shown. The average and standard deviation of four (scUbc6pser) or six (empty plasmid, scUbc6p) independent experiments was calculated and is displayed as a graph. (C) Yeast strain K700 strain was co-transformed with pUB23P, which encodes for the N-end rule substrate Pro-ß-Galactosidase, and with high-copy plasmids coding for scUbc6p or scUbc6pser, or with an empty vector. Equal amounts of cells were labeled with 35S-cysteine/methionine and chased with non-radioactive medium. Aliquots were taken after 0, 20 and 40 minutes chase-time and immunoprecipitated for ß-galactosidase, subjected to SDS-PAGE and analyzed by autoradiography. A typical experiment is shown in the upper part. The average of three independent experiments was calculated and is displayed as a graph.

View larger version (24K): [in a new window]   Fig. 5. (A) Effect of Ubc6p overexpression on mammalian ERAD. The turnover of TCR and F508CFTR was determined in pulsechase experiments with or without expression of wild-type and mutant Ubc6. The calculated half-life of both substrates is summarized in the plot. The turnover was measured upon co-expression of either mmUbc6 or hsUbc6e as indicated. Of each conjugating enzyme a wild-type version (grey bars) or an active site mutant (black bars) was used. As a control, an unrelated protein (GFP; white bars) was expressed. For TCR, data indicated are mean±s.e.m. of three experiments derived from linear regressions of semi-logarithmic transformations of decay kinetics. For F508 CFTR, data are from two representative experiments. Below the plot, representative autoradiograms are shown. (B) Cells stably expressing an unstable cytosolic protein GFPu were transfected with wild-type hsUbc6e or mutant hsUbc6e-C91S, and GFP fluorescence was determined by FACS analysis 2 days later.