Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy.

ABSTRACT

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations ATG autophagy-related; CALCOCO1 calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52 calcium binding and coiled-coil domain protein 2; CLIR MAP1LC3C-interacting region; CQ chloroquine; KO knockout; LIR MAP1LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; MEF mouse embryonic fibroblast; MLN MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR mechanistic target of rapamycin kinase; reticulophagy selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3 TAX1 binding protein 1; ULK unc 51-like autophagy activating kinase; WT wild-type.