UC San Diego

The functions of novel supramolecular structure assembly in enzymes are not fully understood. Analyzing these structures may provide insight into how they regulate enzyme activity as well as any novel roles they serve in the cell. For example, identifying the triggers of GDH1 polymerization could explain how mutations in GLUD1, the human ortholog, cause familial hypoglycemia. Similarly, understanding how PRPS1 filaments form in the nucleus could explain why gain and loss of function mutations have overlapping disease phenotypes. Both PRPS1 and GDH1 were tagged with GFP and HA and transfected into yeast to examine any adverse effects on polymerization. No changes in supramolecular structure integrity were observed in GDH1 strains containing either tag whereas PRPS1 required immunostaining for natural polymerization. GDH3 and GLT1 yeast knockout strains exhibited increased puncta frequency suggesting pathway epistasis. Human PRPS1, HA tagged yeast orthologues PRS3 and PRS5, and PRPS1 superactivity mutations were transformed into yeast to examine the effects on filament formation. PRS3, PRS5, and PRPS1 filaments localized into the cytosol unlike PRPS1 filament formation in the nucleus of human fibroblasts. This suggests that the nuclear localization machinery is unique to mammalian cells. The L129I mutant’s filament frequency and expression did not change. The H193Q mutation led to decreased filament frequency during postdiauxic shift, but no changes in expression level. The A190V, D52H, D183H, and N114S mutations led to both an increase in filament frequency and expression levels. However, all PRPS1 mutants display deformed and enlarged PRPS1 filaments suggesting that they affect filament integrity.