Physical and genetic associations of the Irc20 ubiquitin ligase with Cdc48 and SUMO

Abstract

A considerable percentage of the genome is dedicated to the ubiquitin-proteasome system, with the yeast genome predicted to encode approximately 100 ubiquitin ligases (or E3s), and the human genome predicted to encode more than 600 E3s. The most abundant class of E3s consists of RING finger-containing proteins. Although many insights have been obtained regarding the structure and catalytic mechanism of the E3s, much remains to be learned about the function of the individual E3s. In Chapter I, we characterize IRC20, which encodes a dual RING- and Snf/Swi family ATPase domain-containing protein in yeast that has been implicated in DNA repair. We found that overexpression of IRC20 causes two transcription-associated phenotypes and demonstrate that the Irc20 RING domain possesses ubiquitin E3 activity in vitro. To better understand how Irc20 functions in vivo, two mass spectrometry approaches were undertaken to identify Irc20-associated proteins. Wild-type Irc20 associated with Cdc48, a AAAATPase that serves as an intermediary in the ubiquitin-proteasome system. A second approach using a RING mutant derivative of Irc20 detected increased association of the Irc20 mutant with SUMO. These findings provide a foundation for understanding the roles of Irc20 in transcription and DNA repair.;Understanding E3 ligase function is greatly enhanced by knowledge of its ubiquitylation substrates. Identifying these E3-substrate relationships, however, has proven to be a particularly challenging task. Chapter II describes a genetic approach to systematically screen for candidate genes that potentially function as Irc20 substrates. We identify the nuclear transport karyopherin, SXM1, as an overexpression suppressor of the 2mu IRC20 transcription phenotype. Our attempts to validate Sxml as a ubiquitylation substrate revealed that is not degraded in an IRC20-dependent manner, suggesting that it is not being targeted to the proteasome. Nevertheless, these findings demonstrate a potentially valuable in vivo approach for using overexpression genetics to uncover candidate ubiquitylation substrates of E3 ligases.