Proteasomal Degradation of Rpn4 in Saccharomyces cerevisiae Is Critical for Cell Viability Under Stressed Conditions

Abstract

The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback loop in which the transcription factor Rpn4 induces the proteasome genes and is rapidly degraded by the assembled proteasome. In addition to the proteasome genes, Rpn4 regulates numerous other genes involved in a wide range of cellular pathways. Therefore, the Rpn4-proteasome negative feedback circuit not only controls proteasome abundance, but also gauges the expression of other Rpn4 target genes. Our previous work has shown that Rpn4-induced gene expression is critical for cell viability under stressed conditions. Here we investigate whether proteasomal degradation of Rpn4 is also important for cell survival in response to stress. To this end, we generate a stabilized Rpn4 mutant (Rpn4*) that retains its transcription activity. We find that expression of Rpn4* severely reduces cell viability in response to various genotoxic and proteotoxic agents. This detrimental effect can be eliminated by a point mutation that abolishes the transcription activity of Rpn4*, suggesting that overexpression of some Rpn4 target genes weakens the cell's ability to cope with stress. Moreover, we demonstrate that inhibition of Rpn4 degradation causes synthetic growth defects when combined with proteasome impairment resulting from mutation of a proteasome gene or accumulation of misfolded endoplasmic reticulum membrane proteins. Rpn4 thus represents an important stress-responsive mediator whose degradation as well as availability are critical for cell survival under stressed conditions.

Transcriptome Sequencing (RNA-seq) Analysis of the Effects of Metal Nanoparticle Exposure on the Transcriptome of Chlamydomonas reinhardtii

“…Further work is required to determine whether nTiO 2 , nZnO, and QDs inhibit the proteasome and whether they could be used as cancer chemotherapy agents. This induction of genes encoding proteasome transcripts (Table 4) is similar to the coordinated induction of a battery of genes encoding proteasome subunits in the yeast Saccharomyces cerevisiae and mammalian cells (61)(62)(63)(64). In addition, exposure of S. cerevisiae to nZnO induced three homologous genes regulated by proteasome inhibition (RB60, CDC48, and UFD1) (65).…”

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

“…The protein is extremely unstable, being a substrate for the Ubr2 ligase (Wang et al 2004;Ju et al 2008), and Rpn4 is also degraded by the proteasome in a ubiquitin-independent pathway (Ju and Xie 2006;Ha et al 2012). Consequently, when proteasome function is compromised, Rpn4 levels rise, leading to homeostatic restoration of proteasome activity (Xie and Varshavsky 2001;Metzger and Michaelis 2009;Wang et al 2010). Under conditions of "proteasome stress," proteasomes also exhibit altered composition .…”

“…In many cases, including Gcn4, the transcriptional activation domain was found to overlap with the degradation signal (Salghetti et al 2000). Ubiquitin-dependent proteolysis was thus found to be intimately coupled to the activity of natural and engineered transcription factors (Salghetti et al 2001;Lipford et al 2005;Wang et al 2010). These observations led to the hypothesis that periodic promoter clearance is important for maximal activity.…”

Cuz1/Ynl155w, a Zinc-dependent Ubiquitin-binding Protein, Protects Cells from Metalloid-induced Proteotoxicity

“…Rpn4 recognizes a nine-base sequence in the promoters of these genes, termed the PACE motif (proteasome-associated control element) (14). Moreover, Rpn4 is itself a highly unstable proteasome substrate, creating a homeostatic feedback loop whereby defects in proteasome-mediated degradation result in stabilization of Rpn4, and increased synthesis of ubiquitin-proteasome components until Rpn4 levels are normalized (15)(16). A canonical PACE sequence within the 5Ј-untranslated region of YNL155W is located between residues Ϫ191 and Ϫ183.…”