Characterization of the lysosomal translocation complex for chaperone-mediated autophagy

Abstract

Autophagy is the term used to refer to the intracellular degradation of proteins and organdies in lysosomes. In mammalian cells, there are three main autophagic pathways: Microautophagy, Macroautophagy and Chaperone Mediated Autophagy (CMA). CMA is a selective form of autophagy by which specific cytosolic proteins, bearing a targeting motif, are sent to lysosomes for degradation. The CMA motif is recognized by a cytosolic chaperone, hsc70, which brings the substrate to the lysosome-associated membrane protein type 2A (LAMP-2A), a CMA receptor at the lysosomal surface. Once bound to LAMP-2A, substrate proteins unfold and, assisted by a luminal resident chaperone, cross the lysosomal membrane and are rapidly degraded in the lumen.;The goal of this study was to characterize the series of events that mediate substrate translocation across the lysosomal membrane via CMA and to identify novel regulators of this process.;In the first part, we used four different approaches to characterize the organization of LAMP-2A at the lysosomal membrane. Our studies have revealed that LAMP-2A dynamically transitions among different molecular weight protein complexes at the lysosomal membrane which changes with changes in CMA activity. Our results support that 700kD LAMP-2A complex, favored under conditions associated with higher CMA activity, is required for translocation of cytosolic proteins across the lysosomal membrane via CMA. Instead, substrates only bind to monomeric forms of LAMP-2A, and it is substrate binding that promotes multimerization to form the translocation complex.;In the second, we have identified two novel functions for hsc70 and hsp90 at the lysosomal membrane. We have found that hsc70 induces disassembly of LAMP-2A from the translocation complex when CMA substrates are absent. Binding of Hsp90 to LAMP-2A is required to stabilize it while transitioning through different multimeric complexes.;In the last part, we identified one of the LAMP-2A-interacting partners, GFAP (Glial Fibrillary Acidic Protein) when added to lysosomes enhanced their CMA activity. GFAP binds LAMP-2A in a GTP-dependent manner and we also identified EFlalpha (Elongation Factor 1 alpha) as its associated GTP-binding protein. Preliminary studies with this novel lysosomal GTPase point toward EF1alpha playing a regulatory role in LAMP-2A dynamics.