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


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									Copyright Ó 2010 by the Genetics Society of America
DOI: 10.1534/genetics.109.112227

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

    Xiaogang Wang,*,1 Haiming Xu,*,1,2 Seung-Wook Ha,* Donghong Ju* and Youming Xie*,†,3
                             *Barbara Ann Karmanos Cancer Institute, and †Department of Pathology, Wayne State
                                          University School of Medicine, Detroit, Michigan 48201
                                                    Manuscript received September 24, 2009
                                                  Accepted for publication November 22, 2009

                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.

T   HE Saccharomyces cerevisiae RPN4 gene (also named
     SON1 and UFD5) was originally isolated as a sup-
pressor of sec63-101, a temperature-sensitive mutant of
                                                                           2004, 2007, 2008; Ju and Xie 2004, 2006; Wang et al.
                                                                           2004). Stabilization of Rpn4 by inhibiting the protea-
                                                                           some results in an increase in the expression of the
SEC63, which encodes an essential component of the                         proteasome genes ( Ju et al. 2004; London et al. 2004).
endoplasmic reticulum (ER) translocation channel                           Together, these observations led to a model in which
(Nelson et al. 1993; Johnson et al. 1995; Finley et al.                    the proteasome homeostasis is regulated by a negative
1998; Fujimoro et al. 1998). Subsequent work showed                        feedback circuit. On one hand, Rpn4 upregulates the
that deletion of RPN4 inhibits the degradation of                          proteasome genes; on the other hand, Rpn4 is rapidly
several model substrates of the N-end rule and UFD                         degraded by the assembled/active proteasome. The
(U b fusion degradation) pathways, suggesting the                          Rpn4–proteasome negative feedback circuit provides
involvement of Rpn4 in proteasomal degradation                             an efficient and sensitive means to control the pro-
( Johnson et al. 1995). The underlying mechanism,                          teasome abundance in S. cerevisiae. Whereas the homo-
however, remained unsolved until recent studies                            logs of Rpn4 have not yet been identified in higher
revealed that Rpn4 is a transcription factor for the                       eukaryotes, their proteasome genes are clearly regu-
proteasome genes (Mannhaupt et al. 1999; Xie and                           lated by a negative feedback mechanism (Lundgren
Varshavsky 2001). Interestingly, Rpn4 is an extremely                      et al. 2003; Meiners et al. 2003; Wojcik and DeMartino
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