Abstract
In eukaryotic cells, macroautophagy is a catabolic pathway implicated in the degradation of long-lived proteins and damaged organelles. Although it has been demonstrated that macroautophagy can selectively degrade specific targets, its contribution to the basal turnover of cellular proteins has not been quantified on proteome-wide scales. In this study, we created autophagy-deficient primary human fibroblasts and quantified the resulting changes in basal degradative flux by dynamic proteomics. Our results provide a global comparison of protein half-lives between wild-type and autophagy-deficient cells. The data indicate that in quiescent fibroblasts, macroautophagy contributes to the basal turnover of a substantial fraction of the proteome at varying levels. As contrasting examples, we demonstrate that the proteasome and CCT/TRiC chaperonin are robust substrates of basal autophagy, whereas the ribosome is largely protected under basal conditions. This selectivity may establish a proteostatic feedback mechanism that stabilizes the proteasome and CCT/TRiC when autophagy is inhibited.
Highlights
Within a cell, proteins are in a state of dynamic equilibrium and are continuously synthesized and degraded (Goldberg and St John 1976)
The half-lives of proteins are often intimately linked to their functions, and disruptions in protein degradation have been associated with a number of pathological conditions (Nedelsky et al, 2008)
Creation and Characterization of Autophagy-Deficient Primary Human Fibroblasts In order to establish autophagy-deficient primary cell models, we used clustered regularly interspaced short palindromic repeats (CRISPR) (Cong et al, 2013; Horvath and Barrangou, 2010) to knock out ATG5 and ATG7 in human diploid fibroblasts expressing the catalytic component of human telomerase (HCA2hTert) (Bodnar et al, 1998; Voskarides and Deltas, 2009)
Summary
Proteins are in a state of dynamic equilibrium and are continuously synthesized and degraded (Goldberg and St John 1976). It is generally believed that the UPS is responsible for the degradation of transient shortlived proteins, while autophagy contributes to the degradation of stable long-lived proteins (Ciechanover, 2005). Beyond these general trends, the relative contributions of individual pathways to protein degradative flux have not been quantified on proteome-wide scales. We quantified the relative contribution of autophagy to proteome turnover by comparing protein half-lives between wild-type and autophagy-deficient cells
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