Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy.

hsc-70 (HSPA8) is a cytosolic molecular chaperone， which plays a central role in cellular proteostasis， including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy， chaperone-mediated autophagy， and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein， by combining plasmon resonance， NMR spectroscopy， and amino acid mutagenesis， we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS， and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 μm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.