Assessment of mammalian endosomal microautophagy.

Endosomal microautophagy (eMI) is a type of autophagy that allows for the selective uptake and degradation of cytosolic proteins in late endosome/multi-vesicular bodies (LE/MVB). This process starts with the recognition of a pentapeptide amino acid KFERQ-like targeting motif in the substrate protein by the hsc70 chaperone, which then enables binding and subsequent uptake of the protein into the LE/MVB compartment. The recognition of a KFERQ-like motif by hsc70 is the same initial step in chaperone-mediated autophagy (CMA), a form of selective autophagy that degrades the hsc70-targeted proteins in lysosomes in a LAMP-2A dependent manner. The shared step of substrate recognition by hsc70, originally identified for CMA, makes it now necessary to differentiate between the two pathways. Here, we detail biochemical and imaging-based methods to track eMI activity in vitro with isolated LE/MVBs and in cells in culture using fluorescent reporters and highlight approaches to distinguish whether a protein is a substrate of eMI or CMA.