Functional domains of the Rsp5 ubiquitin-protein ligase

RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase, Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal d omain of approximately 350 amino acids (the beet domain) and a large, nonco nserved aminoterminal domain containing determinants of substrate specifici ty, We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably inte ract with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II ), and the finding that the beet domain, by itself, is sufficient for forma tion of the ubiquitin-thioester intermediate. Mutations within the beet dom ain that affect either the ability to form a ubiquitin-thioester or to cata lyze substrate ubiquitination abrogate in vivo function, strongly suggestin g that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linke d to its essential function. The amino-terminal region of Rsp5 contains thr ee WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not , and requirements for Rpb1 binding and ubiquitination lie within the regio n required for in vivo function. Together, these results support the two-do main model for beet E3 function and indicate that the WW domains play a rol e in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other beet E3 g enes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.