Functional dissection of a HECT ubiquitin E3 ligase.
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.
Lu
Jin-Ying
JY
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Lin
Yu-Yi
YY
Qian
Jiang
J
Tao
Sheng-Ce
SC
Zhu
Jian
J
Pickart
Cecile
C
Zhu
Heng
H
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*
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