Hold on the amputation.

Cargo-based assays have proven invaluable in the study of macroautophagy in yeast and mammalian cells. Proteomic analysis of autolysosomes identified the metabolic enzyme, betaine homocysteine methyltransferase (BHMT), as a potential cargo-based, end-point marker for mammalian macroautophagy. To test whether degradation of BHMT can be used to measure macroautophagic flux in mammalian cells, we created a BHMT fusion protein (GST-BHMT) that demonstrates starvation-induced, site-specific fragmentation in a variety of cell lines. Subcellular fractionation studies show that the GST-BHMT fragment co-fractionates with vesicles containing lysosomal and autolysosomal markers. Furthermore, both pharmacological inhibitors of macroautophagy and depletion of macroautophagy-specific proteins reduce accumulation of the fragment. In the course of these studies, we observed that fragmentation of GST-BHMT did not occur in forms of the reporter with truncation or point mutations that destabilize oligomerization. Since stable oligomerization of BHMT is essential for its catalytic activity, a point mutation known to ablate BHMT activity was tested. We show that accumulation of the GST-BHMT fragment is not impaired in a catalytically inactive mutant, indicating that selective proteolysis of GST-BHMT requires stable quaternary structure independent of effects on activity. Also, the loss of fragmentation observed in the oligomerization deficient mutants does not seem to be due to a defect of sequestration and lysosomal loading, suggesting that disruption of stable quaternary structure affects the ability of a lysosomal protease to cleave the newly-delivered cargo. Finally, we propose that the cargo-based GST-BHMT assay will be a valuable addition to existing macroautophagy assays in mammalian cells.