Macroautophagy-dependent, intralysosomal cleavage of a betaine homocysteine methyltransferase fusion protein requires stable multimerization.
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.
Department of Genome Science, Genome Research Institute, University of Cincinnati, Cincinnati, Ohio 45237, USA.
Carol A
CA
Mercer
Alagammai
A
Kaliappan
Patrick B
PB
Dennis
D000601:Amino Acids, Essential / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D050940:Betaine-Homocysteine S-Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D019281:Dimerization; D004795:Enzyme Stability / Q000502:physiology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000601:Amino Acids, Essential; D016895:Culture Media, Serum-Free; D011993:Recombinant Fusion Proteins; D050940:Betaine-Homocysteine S-Methyltransferase; D005982:Glutathione Transferase