Abstract
Analysis of several Saccharomyces cerevisiae ump mutants with defects in ubiquitin (Ub)-mediated proteolysis yielded insights into the regulation of the polyubiquitin gene UBI4 and of proteasome genes. High-molecular weight Ub–protein conjugates accumulated in ump mutants with impaired proteasome function with a concomitant decrease in the amount of free Ub. In these mutants, transcriptional induction of UBI4 was depending in part on the transcription factor Rpn4. Deletion of UBI4 partially suppressed the growth defects of ump1 mutants, indicating that accumulation of polyubiquitylated proteins is deleterious to cell growth. Transcription of proteasome subunit genes was induced in ump mutants affecting the proteasome, as well as under conditions that mediate DNA damage or the formation of abnormal proteins. This induction required the transcriptional activator Rpn4. Elevated Rpn4 levels in proteasome-deficient mutants or as a response to abnormal proteins were due to increased metabolic stability. Up-regulation of proteasome genes in response to DNA damage, in contrast, is shown to operate via induction of RPN4 transcription.
Highlights
Selective ubiquitin (Ub)-mediated proteolysis (UMP) is the dominating mechanism in the degradation of cytosolic and nuclear proteins in eukaryotic cells [1,2,3]
The proteolytic defects of the ump3-1 mutant were complemented by yeast genomic library plasmids containing the UBI4 gene or the DOA4 gene. ump4-1 is an allele of PRE4 containing a single point mutation leading to an exchange from Ser to Tyr at position 224
An anti-Ub Western blot revealed a less dramatic accumulation of Ub-protein conjugates in the ump1D ubi4-D double mutant in comparison to the ump1-D single mutant (Fig. 1C). These data indicated that an accumulation of Ub-protein conjugates underlies or at least contributes to the growth defects of the ump1-D mutant. Consistent with this assumption, we obtained a partial suppression of the ump1-D growth defects when we introduced the doa4-D mutation (Fig. 1B), which interferes with Ub recycling and reduces the availability of Ub for conjugation
Summary
Selective ubiquitin (Ub)-mediated proteolysis (UMP) is the dominating mechanism in the degradation of cytosolic and nuclear proteins in eukaryotic cells [1,2,3]. These results showed that expression of the other Ub encoding genes UBI1UBI3 is insufficient to maintain a level of free Ub high enough to allow ump3 mutant cells to survive at 37 °C or starvation conditions. Consistent with this, our data showed that cells lacking the Ub-recycling function of Doa4 were unable to maintain normal levels of Ub they showed increased expression of UBI4 compared to wild-type cells (Fig. 1E and F).
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