Macroautophagy-dependent, intralysosomal cleavage of a betaine homocysteine methyltransferase fusion protein requires stable multimerization.
2008
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 stabl...
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