Unravelling chaperone-mediated microautophagy targeting KFERQ-like motif containing proteins in yeast

Under prolonged starvation, mammalian cells activate chaperone-mediated autophagy (CMA) that degrades cellular proteins containing KFERQ-like motifs via lysosomes. During CMA, the lysosomal membrane protein LAMP2A acts as an essential receptor for the HSPA8/HSC70-CMA substrate complex. Thus, the evidence of CMA in organisms lacking LAMP2A on lysosomes/vacuoles is still lacking. Here, we examined the fate of proteins containing such motifs in S. cerevisiae that lack the CMA receptor on vacuoles. Intriguingly, we found that even in the absence of LAMP2A, proteins containing such motifs translocate into vacuoles upon prolonged starvation. We report for the first time that phosphatidylserine acts as an Hsp70-family protein-substrate receptor on the vacuolar membrane to facilitate the substrate translocation into vacuoles. As the newly discovered degradation pathway is dependent upon cytosolic Hsp70 (as in CMA) as well as the ESCRT complex, and involves invagination of the vacuolar membrane, we refer to it as chaperone-mediated microautophagy. Taken together, this study has led to the identification of a novel cellular pathway in S. cerevisiae that facilitates the clearance of cellular proteins under chronic stress.Abbreviations: CMA: chaperone-mediated autophagy; CMA-tag: KFERQ motif; ESCRT: endosomal sorting complexes required for transport; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HSPA8: heat shock protein 8; LAMP2A: lysosomal-associated membrane protein type 2A; Lact-C2: lactadherin C2 domain; PAmCherry: photoactivatable mCherry; PBS: phosphate-buffered saline; PS: phosphatidylserine; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; TBST: Tris-buffered saline with Tween 20.