Figure 3.

ER stress does not impair degradation of Doa10 ERAD-C substrates. A, schematic of Doa10 substrates investigated in this figure. Deg1-Vma12 consists of Deg1, a FLAG (F) epitope, the two-transmembrane protein Vma12, and two copies of the S. aureus protein A (PrA). Deg1-sec62† is Deg1-Sec62 with a point mutation that prevents aberrant translocon engagement, thus rendering the protein a Doa10 substrate. Ub, ubiquitin. B and D, pulse-chase analysis of WT yeast expressing Deg1(*)-Vma12 or Deg1-Sec62(†) cultured in the presence of 10 μg/ml tunicamycin or DMSO for 30 min. Tunicamycin and DMSO were maintained at the same concentration throughout pulse labeling. C, cycloheximide chase analysis of yeast of the indicated genotypes expressing Deg1-Vma12 and Deg1-Sec62 or harboring empty vectors (Vec/Vec), cultured in the presence of 6 mm DTT, 10 μg/ml tunicamycin, or DMSO for 1 h. DTT, tunicamycin, and DMSO were maintained at the same concentration during incubation with cycloheximide. Deg1-Sec62 and Deg1-Vma12 were detected with AlexaFluor-680–conjugated rabbit anti-mouse antibody. Pgk1 served as a loading control. The percentage of Deg1-Vma12 remaining at each time point (normalized to Pgk1) is indicated below the image. Experiments depicted in B and D were performed one time. The experiment depicted in C was performed three times.