Methods to study chaperone-mediated autophagy

Abstract

Chaperone-mediated autophagy (CMA) is a multistep process that involves selective degradation and digestion of a pool of soluble cytosolic proteins in lysosomes. Cytosolic substrates are selectively identified and targeted by chaperones to lysosomes where they are subsequently translocated into the organelle lumen through a dedicated CMA-associated lysosomal membrane receptor/translocation complex. CMA contributes to maintaining a functional proteome, through elimination of altered proteins, and participates in the cellular energetic balance through amino acid recycling. Defective or dysfunctional CMA has been associated with human pathologies such as neurodegeneration, cancer, immunodeficiency or diabetes, increasing the overall interest in methods to monitor this selective autophagic pathway. Here, we describe approaches used to study CMA in different experimental models.

Keywords: Autophagy; Fluorescent reporters; Lysosomes; Organelle isolation; Proteolysis.

Figure 1. Types of mammalian autophagy

Scheme of the basic steps of three different types of mammalian autophagy: Macroautophagy: cargo such as protein aggregates, lipid droplets, organelles, are sequestered into double membrane vesicles (autophagosomes; APH), which then fuse with lysosomes (autophagolysosomes; APL) where cargo degradation occurs. Endosomal microautophagy (e-MI): cytosolic content is delivered “in bulk” or upon interaction with the hsc70 chaperone to the membrane of late endosomes where multivesicular bodies (MVB) form trapping and internalizing the cargo; degradation occurs upon disruption of the vesicles in the endosomal lumen or after fusion of endosomes with lysosomes. Chaperone-Mediated Autophagy (CMA): substrate proteins recognized by hsc70 are targeted to the lysosomes and translocated across the lysosomal membrane assisted by CMA receptor LAMP2A to be degraded by lysosomal hydrolases.

Figure 2. Assays to measure CMA activity

Scheme of the most common assays to measure CMA activity grouped on whether they can be used on cells in culture (top), animal tissues (bottom) or in both (middle). Steady-state assays provide a snap-shot of the status of CMA and can be used for comparative purposes in between conditions. However, complete assessment of CMA requires the use of functional assays as depicted in this scheme.

Figure 3. Discrimination of pathways responsible for intracellular proteolysis

Experimental set up of measurement of total rates of intracellular protein degradation modified to discriminate lysosomal degradation (fraction inhibited by treatment with lysosomal protease inhibitors) from non-lysosomal degradation (remaining upon lysosomal proteolysis blockage). Addition of inhibitors of macroautophagy (i.e. 3-methyladenine) also allows to separate degradation by this pathway (sensitive to 3MA) from degradation by CMA and e-MI (insensitive to 3MA).