Upregulation of the PRB1 gene in the Saccharomyces cerevisiae rim101Delta mutant produces proteolytic artefacts that differentially affect some proteins.
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 rim101Delta 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.
Pérez
Jacqueline
J
Instituto de Microbiología Bioquímica and Departamento de Microbiología y Genética, CSIC/University of Salamanca, Spain.
Gómez
Alberto
A
Roncero
César
C
D016477:Artifacts; D017353:Gene Deletion; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D012097:Repressor Proteins / Q000172:deficiency*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D015854:Up-Regulation
D011480:Protease Inhibitors; C489601:RIM101 protein, S cerevisiae; D012097:Repressor Proteins; C083185:SKT5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae; D012697:Serine Endopeptidases; C023302:yeast proteinase B