The deubiquitinating enzyme Ubp2 modulates Rsp5-dependent Lys63-linked polyubiquitin conjugates in Saccharomyces cerevisiae.
The functions of Lys(63)-linked polyubiquitin chains are poorly understood, as are the enzymes that specifically generate Lys(63)-linked conjugates. Rsp5 is a HECT (homologous to E6AP C terminus) ubiquitin ligase involved in numerous processes, and an associated deubiquitinating enzyme, Ubp2, modulates its activity. A dramatic increase in Lys(63)-linked conjugates was observed in ubp2Delta cells. The formation of these was Rsp5-dependent, and ubp2Delta phenotypes could be suppressed by prevention of formation of Lys(63) conjugates. Cell wall integrity was impaired in rsp5-1 cells and in cells defective in Lys(63)-polyubiquitination, as assayed by calcofluor white sensitivity, and ubp2Delta and rup1Delta mutants suppressed the calcofluor white sensitivity of rsp5-1. A large fraction of the Lys(63) conjugates in ubp2Delta cells bound to Rsp5, and a proteomics approach was used to identify Rsp5 substrates subject to Ubp2 regulation. Two closely related proteins, Csr2 and Ecm21, were among the identified proteins. Both were efficiently Lys(63)-polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. Together, these results indicate that Ubp2 modulates Lys(63)-polyubiquitination of Rsp5 substrates in vivo, including ubiquitination of two newly identified Rsp5 substrates.
Kee
Younghoon
Y
Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712, USA.
Muñoz
William
W
Lyon
Nancy
N
Huibregtse
Jon M
JM
D002473:Cell Wall / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000737:chemistry; D005838:Genotype; D005982:Glutathione Transferase / Q000378:metabolism; D008239:Lysine / Q000737:chemistry*; D009687:Nuclear Proteins; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D025821:Polyubiquitin / Q000737:chemistry*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology; D013696:Temperature; D025801:Ubiquitin / Q000737:chemistry*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism / Q000502:physiology*
C516566:Csr2 protein, S cerevisiae; C422223:ECM2 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D009687:Nuclear Proteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D005982:Glutathione Transferase; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase; C469287:RSP5 protein, S cerevisiae; D008239:Lysine