Sec62p and Sbh1p are N-acetylated. (A) Sec complexes and ribosome-free Sec61 complexes were purified from an SSS1-HA strain by affinity chromatography as described in Methods. Individual bands were excised, trypsin-digested and analyzed by mass spectrometry. N-acetylated proteins are indicated by red asterisks. (B) Mutant sec62-1 cells were transformed with a plasmid expressing wildtype SEC62 or N-acetylation acceptor site mutant sec62-S2Y. SEC62 wildtype cells and untransformed and transformed sec62-1 cells were diluted and growth on solid media monitored at the indicated temperatures. (C) SEC62 wildtype or the indicated mutant cells without or with the indicated plasmids were grown to early exponential phase, cells lysed, proteins separated by SDS-PAGE and prepro-alpha factor (ppaF) detected by immunoblotting.