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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.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms.