FIGURE 2:
Low levels of protein degradation in exponentially growing yeast cells. (A) Schematic representation of target protein localization and function. Proteins are synthesized by ribosomes and enter the ER through the SEC translocon. They are glycosylated by OST (N-glycans) or the PMT complexes (O-glycans). Folding is assisted by chaperones Pdi1p and Kar2p. Further target proteins were the vacuolar protease PrA, the plasma membrane transporter Pdr5p, and the mitochondrial protein Ilv5p. OST subunits are colored green, yellow, or orange according to subcomplexes as proposed previously (Kelleher and Gilmore, 2006). (B) kDEG values for 25 target proteins in wild-type (SMA673) or Δhrd1Δdoa10 (SMA1566) cells. Degradation rates were measured as described in Figure 1A. Error bars represent SD of three biological replicates. *p ≤ 0.05. Turnover of Ost4p could not be determined, since tryptic digest yielded only a 35-residue peptide (out of 36 in total), which was too long to be analyzed by MS accurately. (C) Protein half-lives in whole-cell extracts. Whole-cell extracts of wild type (SMA673) were prepared, and proteins were digested for MS and measured by shotgun LC-MSMS and quantified with MaxQuant. Results of three biological replicates. (D) Proteins with half-lives <12 h (average of three biological replicates) from whole-cell extract in C. The dashed line indicates a half-life of eight cell divisions.