Quality control of protein complex assembly
type: CreativeWork
description: All cells contain numerous multi-subunit protein complexes of exceptional abundance and physiological importance. Prominent examples include the ribosome, proteasome, chaperonin, tubulin, haemoglobin, and many others. A major challenge for the cell is to synthesise individual subunits in the correct stoichiometry. Any imbalance in subunit synthesis results in excess unassembled “orphan” subunits lacking their interaction partner(s). Orphans are accentuated in disease states such as cancer where altered gene expression caused by aneuploidy is common. Acute aneuploidy is known to cause proteotoxicity, probably as a consequence of high orphan burden. How orphan subunits are targeted for degradation to maintain protein homeostasis remains an unanswered question for most of the cell’s major protein complexes. The work described in this thesis investigated how the seven beta subunits of the 20S core proteasome (named PSMB1-7) are targeted for degradation when produced in excess. I used a mammalian *in vitro* translation system to identify candidate interactors of nascent PSMB subunits and found several proteins related to the ubiquitin-proteasome system. Using fluorescent reporters of PSMB subunit degradation in cells depleted of individual candidates, I found that the E3 ubiquitin ligases KCMF1 and UBR4 are required for efficient degradation of unassembled PSMB subunits. Biochemical assays showed that KCMF1 and UBR4 form a stable complex that physically interacts with nascent PSMB subunits. Moreover, using a cell-based flow cytometry assay, I determined which domains of KCMF1 and UBR4 are required for effective degradation of PSMB subunits. Analysis of orphans from other protein complexes showed that the KCMF1-UBR4 complex was also required for efficient degradation of PSMC5 (a 19S proteasomal subunit) and CCT4 (a chaperonin subunit). This was unexpected because orphaned PSMC5 and CCT4 were recently shown to be multiply mono-ubiquitinated by the E3 ubiquitin ligases HERC…
abstract: All cells contain numerous multi-subunit protein complexes of exceptional abundance and physiological importance. Prominent examples include the ribosome, proteasome, chaperonin, tubulin, haemoglobin, and many others. A major challenge for the cell is to synthesise individual subunits in the correct stoichiometry. Any imbalance in subunit synthesis results in excess unassembled “orphan” subunits lacking their interaction partner(s). Orphans are accentuated in disease states such as cancer where altered gene expression caused by aneuploidy is common. Acute aneuploidy is known to cause proteotoxicity, probably as a consequence of high orphan burden. How orphan subunits are targeted for degradation to maintain protein homeostasis remains an unanswered question for most of the cell’s major protein complexes. The work described in this thesis investigated how the seven beta subunits of the 20S core proteasome (named PSMB1-7) are targeted for degradation when produced in excess. I used a mammalian *in vitro* translation system to identify candidate interactors of nascent PSMB subunits and found several proteins related to the ubiquitin-proteasome system. Using fluorescent reporters of PSMB subunit degradation in cells depleted of individual candidates, I found that the E3 ubiquitin ligases KCMF1 and UBR4 are required for efficient degradation of unassembled PSMB subunits. Biochemical assays showed that KCMF1 and UBR4 form a stable complex that physically interacts with nascent PSMB subunits. Moreover, using a cell-based flow cytometry assay, I determined which domains of KCMF1 and UBR4 are required for effective degradation of PSMB subunits. Analysis of orphans from other protein complexes showed that the KCMF1-UBR4 complex was also required for efficient degradation of PSMC5 (a 19S proteasomal subunit) and CCT4 (a chaperonin subunit). This was unexpected because orphaned PSMC5 and CCT4 were recently shown to be multiply mono-ubiquitinated by the E3 ubiquitin ligases HERC…
author: Carrillo Roas, Sara
language: eng
keywords: assembly quality control, protein complex assembly, protein quality control, ubiquitination
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f48cc273-43c7-4498-a48b-5c24b6ae44a6/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2023-11-16
date modified: 2026-07-22T22:24:28Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.106511