Table 1.

Characteristics of a CMA substrate

Characteristic of a CMA substrate	Assay to confirm	Limitations
KFERQ-like motif in its sequence	Sequence analysis	-Not all KFERQ containing proteins are continuously degraded by CMAa -This motif can also target proteins for e-MIb degradation.
Long half-life	Proteolytic assays by metabolic labelling and immunoprecipitation	-More suitable for culture cells. Depends on having antibodies with pull-down capacity.
Detectable in lysosomes	Co-immunofluorescence substrate/ lysosomal markers. Immunoblot in isolate CMA active lysosomes.	-Need to discard endosomal association. -May be degraded too fast to detect it (use protease inhibitors).
Changes in cellular levels or lysosomal association upon CMA modulation	Increase in cellular substrate levels with lysosomal inhibitors or LAMP2Ac genetic reduction. Decreases in substrate levels upon LAMP2A overexpression or treatment with atypical RARd antagonists.	-There are not currently chemical selective inhibitors that allow for rapid blockage of CMA. -Overexpression of LAMP2A to very high level leads to misslocalalization -Blockage of CMA may upregulate degradation by other proteolytic systems.
Interacts with hsc70 in cytosol	Co-immunoprecipitation using cytosol fractions	-Substrate-hsc70f binding is transient and also occurs for e-MI substrates (need to verify interaction with LAMP2A).
Interacts with LAMP2A at the lysosomes	Co-immunoprecipitation using isolated lysosome fractions	-Requires large amount of starting material for lysosomal purification. Interaction with LAMP2A for functional purposes (no degradation) is possible.
Translocates into isolated lysosomes in an ATP/hsc70 dependent manner	In vitro binding and uptake by immunoblot Degradation by intact lysosomes	-Requires purified protein. -Confirming lysosomal integrity is essential to conclude that there is direct uptake.

^aChaperone-mediated autophagy;

^bendosomal microautophagy,

^clysosome-associate membrane protein type 2A;

^dretinoic acid receptor;

^eheat shock cognate protein;