Proteasome-mediated protein degradation is a major regulatory mechanism in the cell. Recent studies from Dr. Xie's laboratory have shown that the proteasome homeostasis in the budding yeast Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 upregulates the proteasome genes and is rapidly degraded by the proteasome. In addition to the proteasome genes, Rpn4 controls the expression of numerous other genes, including a number of key stress response genes. Interestingly, the RPN4 gene itself is induced by a wide variety of environmental stresses. These observations indicate that Rpn4 serves as a hub in a stress response network onto which the environmental stimuli "fan in" and from which the response signals "fan out". The Rpn4-proteasome negative feedback circuit gauges the output signals by keeping the Rpn4 protein level in check and therefore is considered a core regulator of the Rpn4-mediated stress response network. The main focus of Dr. Xie's research is to elucidate the mechanisms underlying Rpn4 degradation. This is a crucial step to understand how the Rpn4-proteasome negative feedback circuit operates in response to environmental stress.
Broader Impacts: This research will make a significant contribution to the training and education of students at various levels. Graduate and undergraduate students and high school students have participated in this study. More graduate students will be recruited to this research. Students will not only receive hands-on training of the powerful techniques of yeast genetics and molecular biology, but also learn how to ask and solve important biological questions. The information obtained from this study will directly benefit protection of the environment.
Proteasome-mediated protein degradation is one of the major cellular processes. Recent studies have shown that the proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 upregulates the proteasome genes and is rapidly degraded by the assembled proteasome. In addition to the proteasome genes, Rpn4 controls a large number of other genes including some key stress response genes encoding factors involved in protein ubiquitylation and DNA repair. Interestingly, the RPN4 gene itself is induced by a wide variety of environmental stresses. These observations suggest that Rpn4 serves as a hub in a stress response network onto which the environmental stimuli "fan in" and from which the response signals "fan out". The Rpn4-proteasome negative feedback circuit conceivably gauges the output signals by keeping the Rpn4 protein level in check and therefore is considered a core regulator of the Rpn4-mediated stress response network. The main focus of this project is to elucidate the mechanisms underlying Rpn4 degradation. This is a crucial step to understand how the Rpn4-proteasome negative feedback circuit operates. Remarkably, Rpn4 can be degraded via two distinct pathways. One is ubiquitin-dependent, whereas the other is ubiquitin-independent. This NSF-supported project aimed to dissect the molecular details of Ub-dependent degradation of Rpn4 (Specific Aim 1) and to explore the mechanism underlying Ub-independent degradation of Rpn4 (Specific Aim 2). The findings resulting from this NSF grant are summarized as follows: 1. The elucidation of the Ub-dependent pathway for Rpn4 degradation We located the Ub-dependent degradation signal (degron) of Rpn4 to an acidic domain consisting of residues 211-229. We found that the degron is regulated by phosphorylation of serine 220. We also identified Ubr2 and Rad6 as the cognate E3 and E2 enzymes for Rpn4 ubiquitylation. In addition, we isolated the MYND-domain protein Mub1 as an essential factor for ubiquitylation of Rpn4. 2. The mapping of the transcription activation domain of Rpn4 We mapped the transcription activation domain of Rpn4 to amino acids 118-210, separate from its degrons. This finding challenges the doctrine that functional overlap of sequences that activate transcription and mediate proteolysis is a common feature of transcription factors. 3. The biological significance of Rpn4 degradation To establish the biological significance of Rpn4 degradation, we created a yeast mutant that expresses a stabilized and yet transcriptionally active version of Rpn4. We showed that this mutant is hypersensitive to a variety of stressors. This work reveals the importance of Rpn4 degradation in stress response. 4. The importance of Rpn4-induced proteasome expression in stress response In addition to the proteasome genes, Rpn4 controls numerous non-proteasome genes involved in stress response. To explicitly examine the role of Rpn4-induced proteasome expression in stress response, we constructed a yeast mutant in which the PRE1 proteasome gene is no longer induced by Rpn4. We showed that this mutant has a much lower proteasome level than its wildtype counterpart and is extremely sensitive to stressed conditions. This finding underscores the importance of Rpn4-regulated proteasome homeostasis in stress response. In addition to the intellectual merits, this study made a significant contribution to the training and education of students at various levels. The trainees participating in this NSF-supported project included one postdoctoral fellow, one graduate student, three undergraduate students, and three high school students. All trainees received excellent training in molecular biology, yeast genetics and biochemical techniques, and learned to critically evaluate data and formulate testable hypotheses. The postdoctoral fellow and the graduate student were able to develop into independent scientists and had the opportunity to mentor undergraduate students and high school students. Undergraduate students and high school students gained hands on experience in the laboratory and were motivated to pursue research careers.
