The two-domain architecture of LAMP2A within the lysosomal lumen regulates its interaction with HSPA8/Hsc70

Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by HSPA8/Hsc70 (heat shock protein 8) are transported into lysosomes for degradation. LAMP2A (lysosomal-associated membrane protein 2A) recruits the substrate/chaperone complex to the lysosome membrane. The structure of LAMP2A comprises a large lumenal domain composed of two homologous subdomains (N-domain and C-domain, both with the β-prism fold), a transmembrane domain, and a short cytoplasmic tail. Although the homophilic interaction between LAMP2A molecules and the interaction between HSPA8 and LAMP2A have been suggested, whether the interactions are direct or mediated by other molecules has remained to be elucidated. We investigated the interactions by using expanded genetic code technologies that generate photo-crosslinking and/or steric hindrance at specified interfaces in mammalian cells. The results suggested that LAMP2A molecules assemble by facing each other with one side of the β-prism in their C-domains. We also detected the photo-crosslinking between the cytoplasmic tail of LAMP2A and HSPA8, revealing this direct interaction. We found that the truncation of the N-domain reduced the amount of HSPA8 that coimmunoprecipitates with LAMP2A. Our present results suggest that the two-domain architecture of the lumenal domains of LAMP2A underlies the interaction with HSPA8 at the cytoplasmic surface of the lysosome.