Characterizing the contribution of chaperone-mediated autophagy to endoplasmic reticulum and lipid droplet homeostasis.
Final Report Abstract
Regulated turnover of proteins is an important factor in cellular and organismal health and survival. In this context, selective autophagy is crucial for the degradation of long-lived proteins and during stress conditions. This study investigated two pathways for selective autophagy, chaperone mediated autophagy (CMA) and endosomal microautophagy (eMi). In both processes, substrates are recognized when the heat shock protein Hsc70 interacts with a pentapeptide motif related to the amino acid sequence KFERQ in the client protein. The lab of Dr. Ana Maria Cuervo has shown that during recovery from unfolded protein stress endoplasmic reticulum (ER) resident chaperones are degraded though CMA and that failure to do so results in cell death. Expanding on these data, I investigated how chaperones are released from the ER lumen into the cytoplasm where they can engage with CMA. Inhibition of the molecular motor driving translocation across the ER membrane prevents accumulation of the ER chaperones in lysosomes. However, further work is required to exclude pleiotropic effects of the inhibitor. Upstream of substrate degradation, the autophagy pathways CMA and eMi share a common mechanism for substrate recognition in the cytosol. To screen for potential cofactors that enable substrate triage between CMA and eMi, I characterized co-chaperones of Hsc70 in late endosomes and lysosomes. This study identifies a member of the BAG domain containing family of Hsc70 nucleotide exchange factors that selectively interacts with Hsc70 in late endosomes but not lysosomes or the cytoplasm. Crucially, blocking this co-chaperone in an in vitro assay shows that it is important for protein degradation through eMi but not CMA. Moreover, reporter cell lines depleted for the co-chaperone display a decrease in eMi and possibly a compensatory increase of CMA activity. Taken together, my analyses strongly indicate that this co-chaperone is a specific regulator of eMi but not CMA. In future studies, these findings will be valuable to differentiate between the two closely related autophagy pathways CMA and eMi and aid in developing selective inhibitors.
