Lysosomal targetomics of mice shows chaperone-mediated autophagy degrades nucleocytosolic acetyl-coA enzymes.

Mice deficient in GHR (growth hormone receptor; KO) have a dramatic lifespan extension and elevated levels of hepatic chaperone-mediated autophagy (CMA). Using quantitative proteomics to identify protein changes in purified liver lysosomes and whole liver lysates, we provide evidence that elevated CMA in KO mice downregulates proteins involved in ribosomal structure, translation initiation and elongation, and nucleocytosolic acetyl-coA production. Following up on these initial proteomics findings, we used a cell culture approach to show that CMA is necessary and sufficient to regulate the abundance of ACLY and ACSS2, the two enzymes that produce nucleocytosolic (but not mitochondrial) acetyl-coA. Inhibition of CMA in NIH3T3 cells has been shown to lead to aberrant accumulation of lipid droplets. We show that this lipid droplet phenotype is rescued by knocking down ACLY or ACSS2, suggesting that CMA regulates lipid droplet formation by controlling ACLY and ACSS2. This evidence leads to a model of how constitutive activation of CMA can shape specific metabolic pathways in long-lived endocrine mutant mice. CMA: chaperone-mediated autophagy; DIA: data-independent acquisition; KO: growth hormone receptor knockout; GO: gene ontology; I-WAT: inguinal white adipose tissue; KFERQ: a consensus sequence resembling Lys-Phe-Glu-Arg-Gln; LAMP2A: lysosomal-associated membrane protein 2A; LC3-I: non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; PBS: phosphate-buffered saline; PI3K: phosphoinositide 3-kinase.