Localization of GAR transformylase in Escherichia coli and mammalian cells

Enzymes of the de novo purine biosynthetic pathway may form a multienzyme c omplex to facilitate substrate flux through the ten serial steps constituti ng the pathway, One likely strategy for complex formation is the use of a s tructural scaffold such as the cytoskeletal network or subcellular membrane of the cell to mediate protein-protein interactions, To ascertain whether this strategy pertains to the de novo purine enzymes, the localization patt ern of the third purine enzyme, glycinamide ribonucleotide transformylase ( GAR Tfase) was monitored in live Escherichia coli and mammalian cells. Gene s encoding human as well as E, coli GAR Tfase fused with green fluorescent protein (GFP) were introduced into their respective cells with regulated ex pression of proteins and localization patterns monitored by using confocal fluorescence microscopy. In both instances images showed proteins to be dif fused throughout the cytoplasm, Thus, GAR Tfase is not localized to an exis ting cellular architecture, so this device is probably not used to concentr ate the members of the pathway, However. discrete clusters of the pathway m ay still exist throughout the cytoplasm.