Abstract
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.
Highlights
Domain [2], whereas RING E3s appear to function as docking surfaces for activated E2s and substrates
Modulation of Lys63 Polyubiquitin Conjugates by Rsp5 and Ubp2—We previously reported that Ubp2 preferentially disassembles Lys63-linked polyubiquitin chains relative to Lys48linked chains in vitro [21]
To determine whether the increased ubiquitin conjugates in the ubp2⌬ cells represented an accumulation of Lys63-linked polyubiquitin chains, we took advantage of strains in which all four endogenous ubiquitin genes were eliminated and replaced by a plasmid-borne wild type ubiquitin gene or a mutated ubiquitin gene encoding K63R ubiquitin [25]
Summary
MATa his leu met ura MAT␣ rsp ubp2⌬::KanMX6 his4-912 ␦R5 lys2-128⌬ ura MAT␣ rsp rup1⌬::KanMX6 his4-912 ␦R5 lys2-128⌬ ura MATa ubp2⌬::KanMX6 his leu met ura MATa lys801 leu 112 ura his3-⌬ 200 trp1-1[am] ubi1-⌬1::TRP1 ubi2-⌬2::ura ubi3-⌬ ub-2 ubi4-⌬. Isogenic to YK018 except for pUB197 instead of pUB39 MAT␣ rsp csr2⌬::KanMX6 his4-912 ␦R5 lys2-128⌬ ura MAT␣ rsp ecm21⌬::KanMX6 his4-912 ␦R5 lys2-128⌬ ura MATa ubp2⌬::KanMX6 rup1⌬::HIS3 leu met ura MATa ubp3⌬::KanMX6 his leu met ura MATa ubp4⌬::KanMX6 his leu met ura. Lys48-linked polyubiquitin chains are the primary signals for targeting to the 26 S proteasome, whereas monoubiquitination and some other forms of polyubiquitin chains appear to mediate alternative functions [22,23,24]. Lys63-linked chains serve non-proteolytic functions in DNA repair pathways [25,26,27], kinase activation [28], and receptor endocytosis [6, 7], they may be capable of proteasome targeting [29]. We show that Rsp and Ubp modulate Lys63-linked polyubiquitination in vivo, and the essential function of Rsp at elevated temperature requires Lys polyubiquitination and identify two new targets of Rsp and Ubp that are subject to Lys ubiquitination
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