I would like to suggest cathepsin A and LAMP2A as reliable markers of chaperon-mediated autophagy.

Ref.

EMBO J. 2003 Jan 2;22(1):47-59.

Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor.

Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of unknown physiological function. We now demonstrate that this protease activity triggers the degradation of the lysosome-associated membrane protein type 2a (lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of lamp2a is important because its level in the lysosomal membrane is a rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a degradation, higher levels of lamp2a and higher rates of CMA. Restoration of PPCA protease activity increases rates of lamp2a degradation, reduces levels of lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the lysosomal membrane and cleaves lamp2a near the boundary between the luminal and transmembrane domains. In addition to the well-studied role of PPCA in targeting and protecting two lysosomal glycosidases, we have defined a role for the proteolytic activity of this multifunctional protein.

Go J Yoshida Thank you so much for your reply. Cathepsin A is a new addition to the bucket. Thanks for sharing this reference.

I understand among all, HSC70 and LAMP2a are the widely accepted markers but Can you comment over using HSP70 and LAMP1 as CMA markers?

Tagging or a known autophagy substrate, or recording its delivery to a lysosome is one of the most robust ways to measure autophagy.

Measuring the delivery of a substrate to the lysosome is unambiguous; it is, by definition autophagy.

Fluorescent protein based tags like Keima and Rosella allow you to quantify and measure the delivery of your substrate to a lysosome. This might not work for CMA though, since the substrates are unfolded on lysosomal entry, and FPs need to be folded to fluoresce...

... here's a free idea for you though; what if you attach a FLAG or HA tag to a bunch of KFERQ-like motifs? heres a few reasons why I suggest it:

1) Its delivery to the lysosome via CMA would break down the tag and allow you to probe the total levels by western blot.

2) You'd able to use immunoflourescence to quantify and image the overlap between the KFERQ fusion protein and the lysosomes (a good secondary metric).

3) It's not endogenous, so theres no ambiguity in terms of pleitropic effects influencing expression.

Hope that helps, Let me know what you think.

Lysosomal levels of LAMP-2A and lysosomal-hsc70 increase with the increase in CMA activity (Agarraberes et al., 1997; Cuervo and Dice, 2000). Increase in CMA activity can be assayed via immunoblot for LAMP-2A and lys-hsc70 in lysosomes isolated from the tissues/cells of interest. Note that total cellular levels of LAMP-2A and hsc70 often remain constant as most of the changes occur in the particular group of lysosomes involved in CMA (approximately 30–60% of total lysosomes depending on cell type and cell conditions). it is advisable, when possible, to analyze the levels of LAMP-2A in the lysosomal fraction. Protocol for the isolation of lysosomes with high and low CMA activity from tissue samples can be found in Cuervo et al., 1997.

Colocalization of LAMP-2A and hsc70 by immunofluorescence is used often to identify the CMA-active lysosomes in cultured cells. This procedure can be performed following a standard immunofluorescence protocol, with the exception that fixation of the samples should be done using methanol. Methanol fixation will eliminate the soluble form of hsc70 (very abundant in the cytosol) allowing for the detection of the low percentage vesicle-associated hsc70.

Although Hsc70 and Hsp70 are highly homologous (86% identity in amino acid sequence), only Hsc70 but not Hsp70 has been implicated in mediating CMA as a molecular chaperone that stimulates cargo protein translocation across membrane (Bandyopadhyay and Cuervo, 2008).