Our long-term goal is to understand how the fate of proteins is regulated by the ubiquitin (Ub) system. Ub, an abundant 76-residue protein, is highly conserved among eukaryotes. Ubiquitylation - the covalent conjugation of Ub to lysine residues on other intracellular proteins - regulates a myriad of cellular processes, including cell cycle progression, DNA repair, transcription, stress responses and signal transduction. Ub is best known as a signal to target proteins for destruction by a multisubunit, ATP dependent protease termed the proteasome. How the substrates are delivered to the proteasome is one of the most challenging issues in the field. It is proposed that adaptor molecules, which selectively recognize ubiquitylated substrates, perform this vital function in deciding the final destination of substrates. We will focus on S. cerevisiae Rad23, a candidate adaptor molecule involved in delivering ubiquitylated substrates to the proteasome. Rad23 has two functional domains: a ubiquitin-like element (UBL), and a ubiquitin-associated motif (UBA). The UBL motif was shown to directly bind the proteasome subunit Rpn1. Several groups including ours found that the UBA domain preferentially binds ubiquitylated substrates. And yeast cells lacking Rad23 are deficient in proteolysis. Importantly, Rad23 promotes the formation of the proteasome-Ub conjugates complex in vivo and in vitro. More recently, we found that Rad23 and Dsk2 interact with Ufd2, an E4 enzyme important for Ub-chain assembly. Based on biochemical properties and genetic evidence, we propose that Rad23-like adaptor proteins recognize multi-ubiquitylated substrates and deliver them to the proteasome through various binding partners. The yeast Ufd2-Rad23 complex regulates the degradation of UFD substrates, Hmg-CoA reductase, and the transcription factor Spt23, and prion protein. We propose the following aims in an effort to decipher the biological role of the adaptor molecules in substrate proteolysis.
Aim 1 is to understand the mechanism underlying the substrate selectivity of Rad23.
Aim 2 is to determine the regulation of the Rad23-Ufd2 complex.
Aim 3 is to define the function of Rad23-mediated proteolysis in prion biogenesis. These studies should reveal novel insights into the mechanisms and functions of the Ub system, and provide defined molecular targets for future intervention in human diseases. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Membrane Biology and Protein Processing (MBPP)
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Shapiro, Bert I
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University of Texas Health Science Center San Antonio
Other Basic Sciences
Schools of Medicine
San Antonio
United States
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Cheng, Haili; Bao, Xin; Rao, Hai (2016) The F-box Protein Rcy1 Is Involved in the Degradation of Histone H3 Variant Cse4 and Genome Maintenance. J Biol Chem 291:10372-7
Bao, Xin; Johnson, Jill L; Rao, Hai (2015) Rad25 protein is targeted for degradation by the Ubc4-Ufd4 pathway. J Biol Chem 290:8606-12
Shao, Jia; Choe, Vitnary; Cheng, Haili et al. (2014) Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation. PLoS One 9:e92290
Krzeszinski, Jing Yan; Choe, Vitnary; Shao, Jia et al. (2014) XPC promotes MDM2-mediated degradation of the p53 tumor suppressor. Mol Biol Cell 25:213-21
Baek, Guem Hee; Cheng, Haili; Kim, Ikjin et al. (2012) The Cdc48 protein and its cofactor Vms1 are involved in Cdc13 protein degradation. J Biol Chem 287:26788-95
Baek, Guem Hee; Kim, Ikjin; Rao, Hai (2011) The Cdc48 ATPase modulates the interaction between two proteolytic factors Ufd2 and Rad23. Proc Natl Acad Sci U S A 108:13558-63
Liu, Chang; van Dyk, Dewald; Choe, Vitnary et al. (2011) Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase. J Biol Chem 286:43660-7
Liu, Chang; van Dyk, Dewald; Xu, Ping et al. (2010) Ubiquitin chain elongation enzyme Ufd2 regulates a subset of Doa10 substrates. J Biol Chem 285:10265-72
Liu, Chang; Choe, Vitnary; Rao, Hai (2010) Genome-wide approaches to systematically identify substrates of the ubiquitin-proteasome pathway. Trends Biotechnol 28:461-7
Yan, Jing; Zhang, Di; Di, Yujun et al. (2010) A newly identified Pirh2 substrate SCYL1-BP1 can bind to MDM2 and accelerate MDM2 self-ubiquitination. FEBS Lett 584:3275-8

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