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.

Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

“…However, the amount of proteins that are single ubiquitinated was not assessed in this case. A different study also performed in yeast showed that 56% of all ubiquitin modified proteins are either monoubiquitinated or have an end-cap ubiquitin, and the remaining bulk is given to polyubiquitin modified proteins (Ziv et al, 2011). In this case the relative abundance of different types of polyubiquitin chains was quite different from the early observations in yeast: the K48 polyubiquitin chains were once again the most prevalent ones (21%), followed by K63 (18%), K29 (5%), K11 (0.6%), K33 ($0.1%) and K27 ($0.1%), while K6 was not detected (Ziv et al, 2011).…”

Deubiquitylases From Genes to Organism

“…Proteomic studies reveal that each of the isopeptide linkage types is represented to a significant extent in both yeast and mammalian cells (54,117,196,282,298). The attachment of ubiquitin chains with any isopeptide linkage excepting Lys63 appears to target substrates to the proteasome in vivo (54,172).…”

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae

“…Cell extracts were prepared from log-phase cells using a TCA lysis method (Ziv et al 2011). Eighty micrograms of total protein were loaded on SDS-PAGE.…”

“…The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hr, heavy and light cells were harvested and lysed using a previously described trichloroacetic acid (TCA) lysis method (Ziv et al 2011). To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild-type yeast cells were exposed to 0.01% MMS for 3 hr, and the (H)-SILAC-labeled wild-type yeast cells were mock-exposed.…”