טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZiv-Uziel Inbal
SubjectProteomic Investigation of the Conjugated Ubiquitin
Landscape
DepartmentDepartment of Biology
Supervisor Professor Michael Glickman
Full Thesis textFull thesis text - English Version


Abstract

Protein ubiquitination through lysine residues regulates many crucial cellular processes. The ubiquitin molecule itself contains seven lysines which can all 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 non-extended, 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. Despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery ‑ the ubiquitin-proteasome system (UPS) and the ubiquitin trafficking system (UTS) ‑ were unevenly perturbed by expression of K0 ubiquitin.

Targeting to the 26S proteasome is an essential outcome of ubiquitination. Several proteins which bind polyUb have been proposed to act as shuttles, delivering ubiquitinated substrates to the proteasome. We have characterized the relationship between two such shuttles, Rpn10 and Dsk2, and ubiquitin. A hierarchy of affinities emerged with Dsk2 binding monoubiquitin tighter than Rpn10 did, whereas Rpn10 preferred the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin-chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. In vivo analysis revealed that by masking its UBL domain, Rpn10 filters Dsk2 interactions, maintaining proper balance of the ubiquitin-proteasome system.