title: Functional dissection of a HECT ubiquitin E3 ligase.
abstract: Ubiquitination is one of the most prevalent protein post-translational modifications in eukaryotes, and its malfunction is associated with a variety of human diseases. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitination remain largely unknown. Here we used a combination of yeast proteome chip assays, genetic screening, and in vitro/in vivo biochemical analyses to identify and characterize eight novel in vivo substrates of the ubiquitinating enzyme Rsp5, a homolog of the human ubiquitin-ligating enzyme Nedd4, in yeast. Our analysis of the effects of a deubiquitinating enzyme, Ubp2, demonstrated that an accumulation of Lys-63-linked polyubiquitin chains results in processed forms of two substrates, Sla1 and Ygr068c. Finally we showed that the localization of another newly identified substrate, Rnr2, is Rsp5-dependent. We believe that our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Jin-Ying
authors.initials: JY
authors.lastname: Lu
authors.forename: Yu-Yi
authors.initials: YY
authors.lastname: Lin
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.forename: Sheng-Ce
authors.initials: SC
authors.lastname: Tao
authors.forename: Jian
authors.initials: J
authors.lastname: Zhu
authors.forename: Cecile
authors.initials: C
authors.lastname: Pickart
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
mesh_terms: D010450:Endopeptidases / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D008239:Lysine / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D025821:Polyubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase; C469287:RSP5 protein, S cerevisiae; D008239:Lysine