THE GST-BHMTASSAY AND RELATED ASSAYS FOR AUTOPHAGY

The endpoint of the autophagic process is the breakdown of delivered cytoplasmic cargo in lysosomes. Therefore, assays based on degradation of cargo are of particular interest in that they can measure regulation of the entire autophagic process, including changes in cargo delivery and breakdown in the lytic compartment. Betaine homocysteine methyltransferase (BHMT) is one of many cytosolic proteins found in the mammalian autophagosome, and delivery of BHMT to the lysosome results in its proteolysis to discrete fragments under certain conditions. Making use of these observations, the GST-BHMT assay was developed as an endpoint, cargo-based autophagy assay. Using this assay as a starting point, additional cargo-based assays have been developed with the potential to measure autophagic degradation of specific subcellular compartments. Here we describe the development and validation of these assays.