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. Author manuscript; available in PMC: 2011 May 12.
Published in final edited form as: Cell. 2010 Nov 12;143(4):579–591. doi: 10.1016/j.cell.2010.10.028

Figure 2. Usa1p-mediated oligomerization of Hrd1p is required for ERAD-L.

Figure 2

(A) Hrd1p-HA and Hrd1p-Myc were co-expressed under the endogenous promoter in either wild type (wt) or usaΔ cells. Detergent-solubilized membranes were treated with the bifunctional crosslinkers DSS or EGS, as indicated. Following quenching of the crosslinking reaction, immunoprecipitation with HA-antibodies was performed. Bound proteins were analyzed by SDS-PAGE followed by immunoblotting with Myc or Usa1p antibodies. The arrowhead indicates the position of a crosslinked species containing both Hrd1p and Usa1p.

(B) Hrd1p-HA and Hrd1p-Myc were co-expressed in cells containing either wild type Usa1p or Usa1p mutants with the indicated deletions. Detergent-solubilized membranes were subjected to immunoprecipitation (IP) with HA-antibodies and bound proteins were analyzed by SDS-PAGE and immunoblotting with HA-, Myc-, or Usa1p- antibodies. H and U indicate the segments in Usa1p that are responsible for interaction with Hrd1p and Usa1p, respectively.

(C) Usa1p-HA was co-expressed with FLAG-Usa1p or with the indicated FLAG-tagged deletion mutants of Usa1p. Detergent-solubilized membranes were subjected to immunoprecipitation (IP) with HA-antibodies and bound proteins were analyzed by SDS-PAGE and immunoblotting with HA- or FLAG- antibodies. Lanes 5 and 8 and the corresponding lanes 13 and 16 show the results with two independent clones of the same construct.

(D) Hrd1p-HA and Hrd1p-Myc or Hrd1(C399S)-HA and Hrd1(C399S)-Myc were co-expressed in cells containing or lacking Usa1p. Detergent-solubilized membranes were subjected to immunoprecipitation (IP) with HA-antibodies and bound proteins were analyzed by SDS-PAGE and immunoblotting with HA- or Myc- antibodies. For cells lacking Usa1p, two independent clones co-expressing Hrd1p-HA and Hrd1p-Myc (Lanes 5 and 6 and the corresponding lanes 13 and 14) or Hrd1(C399S)-HA and Hrd1(C399S)-Myc (Lanes 7 and 8 and the corresponding lanes 15 and 16) are shown.

(E) Kinetics of CPY*-HA degradation in cells expressing either wild type Usa1p or the indicated deletion mutants. The levels of CPY*-HA were determined by immunoblotting at different time points after cycloheximide addition (Figure S3). Shown are the means and standard deviations of three independent experiments.

See also Figure S3.