Table 2.
The CMA toolkit: overview of the approaches that can be used to identify CMA substrates
| Substrate feature | Description of the assay | Additional notes and features |
|---|---|---|
| Presence of KFERQ motif | • Scan for the motif in the amino acid sequence of the protein of interest • Incomplete motifs could be converted to a complete motif by post-translational modifications (Box 1) |
Also present in eMI substrates |
| Association with lysosomes | • Colocalization with CMA-active lysosomes (HSC70+) in intact cells • Immunoblot in isolated lysosomes to demonstrate preferential association with CMA-active lysosomes |
Proteins can associate with lysosomes to be functional |
| Degradation in lysosomes | Increased association of the protein of interest with CMA-active lysosomes upon lysosomal inhibition assayed by either immunofluorescence in intact cells or immunoblot of isolated lysosomes | Levels of substrates for other autophagic pathways also increase |
| Change in protein levels and degradation when CMA activity is modified | • Increased levels of the protein of interest or reduced degradation rates upon blockage of CMA (knockdown or knockout for LAMP2A) • Reduced levels of the protein of interest or increased degradation rates upon activation of CMA (overexpression of LAMP2A or AR7 compound) |
Compensatory upregulation of other forms of autophagy might mask this effect |
| Interaction with CMA components | Coimmunoprecipitation of the protein of interest with HSC70 in cytosolic fractions or LAMP2A in lysosomal fractions | Binding to HSC70 is not a sufficient criterion as it can occur because of unfolding or targeting to other pathways |
| Lysosomal uptake | In vitro assay with intact isolated lysosomes pretreated or not with protease inhibitors incubated with the protein of interest | Most conclusive proof of being a CMA substrate |
For comprehensive details on assays and methodology to validate a protein as a CMA substrate, see REF.117. CMA, chaperone-mediated autophagy; eMI, endosomal microautophagy; HSC70, heat shock cognate 71 kDa protein (also known as HSPA8); LAMP2A, lysosome-associated membrane protein type 2A.