title: A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis.
abstract: Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.
authors.affiliation: Department of Biology, Technion Israel institute of Technology, Haifa 32000, Israel.
authors.forename: Inbal
authors.initials: I
authors.lastname: Ziv
authors.forename: Yulia
authors.initials: Y
authors.lastname: Matiuhin
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.forename: Zoi
authors.initials: Z
authors.lastname: Erpapazoglou
authors.forename: Sebastien
authors.initials: S
authors.lastname: Leon
authors.forename: Marina
authors.initials: M
authors.lastname: Pantazopoulou
authors.forename: Woong
authors.initials: W
authors.lastname: Kim
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.forename: Rosine
authors.initials: R
authors.lastname: Haguenauer-Tsapis
authors.forename: Noa
authors.initials: N
authors.lastname: Reis
authors.forename: Michael H
authors.initials: MH
authors.lastname: Glickman
authors.forename: Oded
authors.initials: O
authors.lastname: Kleifeld
mesh_terms: D008239:Lysine / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D020125:Mutation, Missense; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021381:Protein Transport; D020543:Proteome / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D050505:Mutant Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D046988:Proteasome Endopeptidase Complex; D008239:Lysine