Upregulation of the PRB1 gene in the Saccharomyces cerevisiae rim101Δ mutant produces proteolytic artefacts that differentially affect some proteins

Abstract

Proteolytic degradation during protein processing in yeast is usually prevented by the addition of protease inhibitors or strict cooling of the samples. In this report we show that, while these precautions are sufficient for some strains, they are clearly insufficient for others. Specifically, we show that the stability of some proteins, such as Slt2p or Chs4p, but not others, is severely compromised in the rim101 mutant due to the upregulation of the PRB1 gene, which leads to higher levels of proteinase B activity. This degradation can be almost completely prevented by an overdose of subtilisin-like protease inhibitors, such as PMSF, or by avoiding cell freezing. Growth under other conditions that increase proteinase B activity also leads to the differential degradation of some proteins. Here, analysis of several commercial protease inhibitor cocktails indicated that all of them lacked enough subtilisin-like protease inhibitors to prevent any excess of proteinase B activity. Therefore, much stricter experimental protocols than those routinely used are necessary to prevent the artefactual interpretation of protein levels in strains or conditions that increase proteinase B activity.

N-Terminal α7 Deletion of the Proteasome 20S Core Particle Substitutes for Yeast PI31 Function

“…Peptidase inhibitors were required to inhibit the processing of TD133 proteasome subunits by upregulated Prb1 during the purification procedure (31). For the preparation of CPs used in Fig.…”