A co-translational ubiquitination pathway for quality control of newly synthesized proteins

Date

2014-08

Authors

Wang, Feng, Ph. D.

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Abstract

Previous studies indicated that 6%-30% of newly synthesized proteins are rapidly degraded by the ubiquitin-proteasome system. This has generally been assumed to occur post-translationally, following failure of chaperone-assisted folding mechanisms. However, the extent and significance of co-translational quality control remains largely unknown. In investigations of ISG15, an interferon-induced ubiquitin-like protein, our lab found that ISG15 is conjugated to a very broad spectrum of newly synthesized proteins. The major ligase for ISG15, Herc5, co-fractionated with polysomes, and further studies indicated that the processes of translation and ISGylation were closely coupled. Here, I employ an in vitro run-off translation system and puromycin labeling experiments to demonstrate that nascent polypeptides are ISGylated within active translation complexes, providing direct support for the co-translational mechanism for ISG15 conjugation. Approaches developed for studying co-translational ISGylation were subsequently used to examine co-translational ubiquitination (CTU), which we hypothesized might be important in quality control of newly synthesized proteins. Consistent with this, I found that the pathway for degradation of newly synthesized proteins was initiated while proteins were being translated, with ubiquitination of actively translating nascent polypeptides. CTU is a conserved and robust pathway from yeast to mammals, with 5-6% of total nascent polypeptides being ubiquitinated in S. cerevisiae, and 12-15% in human cells. CTU products contained primarily K48-linked polyubiquitin chains, consistent with a proteasomal targeting function. Although nascent chains previously have been shown to be ubiquitinated within stalled and defective translation complexes (referred to here as CTU [superscript S]), nascent chain ubiquitination also occurred within active translation complexes (CTU [superscript A]). CTU [superscript A] accounted for approximately two-thirds of total CTU (CTU[superscript T]) in human cells and approximately half of CTU[superscript T] in yeast cells. CTU[superscript A] was increased in response to agents that induce protein misfolding, whereas CTU[superscript S] was increased in response to agents that led to translational misreading or stalling. These results indicate that ubiquitination of nascent chains occurs in two contexts and define CTU[superscript A] as a component of a quality control system that marks proteins for destruction before their synthesis is complete. Finally, decreased translation fidelity is thought to lead to the accumulation of misfolded proteins and hasten the aging process. As CTU is a pathway for quality control of newly synthesized proteins, I explored whether CTU plays a protective role during the replicative aging process in budding yeast. Consistent with previous reports using human cells, I found that newly synthesized proteins are a major source of proteasome substrates under non-stressed conditions. Transient proteasome inhibition (using MG132) led to a decrease of yeast replicative life span (RLS), whereas simultaneous treatment with cycloheximide, a translation inhibitor, suppressed this effect. Deletion of Ltn1, the major E3 ligase of the CTU[superscript S] pathway, also shortened the RLS of yeast. Together, these results provide a preliminary set of evidence supporting the hypothesis that the quality of newly synthesized proteins is an important determinant of aging.

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