ABSTRACT
In eukaryotic cells, the macroautophagy pathway has been 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 had previously not been quantified on proteome-wide scales. In a recent study, we utilized dynamic proteomics to provide a global comparison of protein half-lives between wild-type and autophagy-deficient cells. Our results indicated that in quiescent fibroblasts, macroautophagy contributes to the basal turnover of a substantial fraction of the proteome. However, the contribution of macroautophagy to constitutive protein turnover is variable within the proteome. The methodology outlined in the study provides a global strategy for quantifying the selectivity of basal macroautophagy.
KEYWORDS: basal autophagy, CCT/TRiC, degradative flux, proteasome, proteomics, ribosome, selective autophagy, turnover
Within a cell, proteins are in a state of dynamic equilibrium and are continuously synthesized and degraded. Protein turnover is one of the most metabolically expensive processes in the human body, consuming as much as 20% of total energy expenditure in a resting adult. The recycling of proteins plays 2 critical roles in vivo. First, it ensures that proteins are able to establish new steady levels following changes in their synthesis rates. Second, it acts as a quality control mechanism by clearing proteins that have been damaged by irreversible covalent modifications or misfolding. Rates of turnover are highly variable between proteins and can range from a few minutes to several years. Kinetics of turnover are often intimately linked to functions of proteins. For example, signaling proteins tend to have short half-lives in order to rapidly achieve altered expression levels, whereas abundant housekeeping proteins are typically more stable in order to reduce cellular energy expenditure associated with protein synthesis.
The constitutive turnover rate of a protein is established by its propensity to be cleared by a number of different degradation pathways. In eukaryotic cells, major degradation pathways include the ubiquitin-proteasome system (UPS), lysosomal pathways such as macroautophagy, endosomal degradation and ER-associated degradation, and processes involving numerous substrate-specific cytosolic and organelle-resident proteases. Although the degradation route of a protein is often limited by its subcellular localization, most proteins have the potential to be degraded by multiple orthogonal degradation pathways. For example, in eukaryotic cells, cytoplasmic proteins can potentially be degraded by both the macroautophagy and UPS pathways.
Despite much recent progress, the cellular logic and mechanistic basis of the division of labor between different cellular protein degradation pathways remains incompletely understood. For example, the relative contribution of the major degradation pathways to the basal turnover of individual proteins within the human proteome has not been quantified and the molecular basis of selectivity for each pathway remains under active investigation.
In a recent study, we quantified the relative contribution of macroautophagy to the basal turnover of proteins in quiescent human fibroblasts. In order to analyze the selectivity of basal autophagy on a global scale, we employed isotopic labeling and mass spectrometry to measure changes in the turnover kinetics of the proteome induced by CRISPR-mediated deletion of 2 genes required for canonical macroautophagy: ATG5 and ATG7. This approach took advantage of recently developed dynamic proteomic methodologies and analysis tools that allow precise quantification of turnover kinetics on proteome-wide scales. Our data enabled us to analyze the impact of autophagy inhibition on the basal turnover and half-lives of more than 4,000 proteins in quiescent human fibroblasts.
The study highlighted a number of principles regarding the selectivity of canonical macroautophagy. A considerable fraction of the proteome can be degraded, to different extents, by both ATG5- and ATG7-dependent and -independent degradation pathways. However, whereas short-lived proteins are degraded almost exclusively by ATG5- and ATG7-independent pathways, long-lived proteins can be robustly degraded by both ATG5- and ATG7-dependent and -independent pathways. Indeed, even among the most stable proteins, ATG5- and ATG7-dependent autophagy typically accounted for less than 50% of basal protein flux.
Our study also indicated that there are clear biases in basal autophagic target selection within the proteome. For example, we showed that the proteasome and the CCT/TRiC chaperonin complexes are robustly targeted for macroautophagy (more than 20% of their basal degradative flux occurs through ATG5- and ATG7-dependent pathways) whereas the ribosome is protected from autophagy under basal conditions. The latter observation was unexpected since ribosomes are well-documented targets of selective autophagy under starvation conditions. The results suggest that under basal and starvation conditions ribosomes may harbor differing modifications that modulate their susceptibility to autophagic degradation.
The fact that the proteasome is targeted for basal autophagy whereas the ribosome is largely protected, effectively establishes a self-compensatory system of protein degradation and synthesis. Under conditions where basal autophagy is inhibited, the proteasome complex is stabilized and the rate of degradative flux through the UPS is enhanced. Conversely, ribosomes are excluded from basal autophagy and do not accumulate when autophagy is inhibited. This ensures that protein synthesis does not increase and exacerbate the accumulation of proteins under conditions where autophagic clearance is already compromised. Additionally, the stabilization of chaperones may counteract proteostatic disruptions resulting from the inhibition of autophagy.
Our study raises a number of questions regarding the selectivity of basal autophagy. First, what are the molecular mechanisms that determine the susceptibility of a given protein to basal autophagy? Recent studies have identified a number of autophagy receptors that play critical roles in the recognition and delivery of cargos to the developing autophagosome for engulfment and subsequent lysosomal degradation. The observed selectivity of basal autophagy may thus reflect the differential affinity of proteins toward this set of receptors or perhaps the presence of a larger compendium of cargo-specific autophagic receptors within the cell that have yet to be identified.
Second, are protein subpopulations destined for basal degradation by different pathways biochemically distinct or are degradation routes established stochastically? Our studies indicate that even for long-lived proteins, only a fraction of the population is degraded by basal autophagy. For example, we estimate that ∼25% of the basal turnover flux of the TRiC/CCT occurs through autophagy, whereas the remainder occurs through alternative degradation pathways. It is unclear whether the population that is degraded through autophagy is stochastically selected, or whether it encompasses a subpopulation that is biochemically distinct. For example, given that autophagy has the capacity to target damaged proteins, and that protein damage is accumulated gradually over time, it may be expected that the subset of the proteome that is turned over by autophagy may be particularly enriched in aged and damaged proteins.
Last, whether the magnitude and selectivity of basal autophagic flux measured in our study is specific to quiescent fibroblasts or can be extended to other cell types and environmental conditions remains to be determined. We think that the proteomic approach outlined in our study may provide a powerful global approach for elucidating the mechanisms of autophagic selectivity in diverse cell types and environmental conditions.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Funding
Funding is provided by the National Science Foundation (NSF), grant number MCB-1350165.