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 domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three 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 region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role 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 hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.
Wang
G
G
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1059, USA.
Yang
J
J
Huibregtse
J M
JM
D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D056827:Endosomal Sorting Complexes Required for Transport; D020104:Immunophilins / Q000378:metabolism; D008025:Ligases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D022021:Tacrolimus Binding Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D008025:Ligases; C469287:RSP5 protein, S cerevisiae