Transformylase Enzymes Dihydrofolate Reductase

Project Summary/Abstract Combining isotopic labeling and gas cluster ion beam surface ionization mass spectrometry (GCIB-SIMS), we found the purinosome metabolon consists of nine enzymes that channel phosphoribosyl pyrophosphate to AMP and GMP. The metabolon greatly increases the pathway flux and unexpectedly favors partitioning of IMP to AMP. Chemical imaging of the cell revealed “hot spots” of these metabolites. We presume that these are located at the microtubule/mitochondrion interface based on temporal fluorescent imaging that found a directional movement of the purinosome along the microtubules to the mitochondria. We uncovered two mechanisms that govern purinosome assembly. One involves the participation of the chaperones HSP90/70 that act to fold two nascent enzymes prior to their incorporation into the purinosome. A second involves the multiple phosphorylation that occurs on the majority of the pathway enzymes upon purinosome assembly. We plan to exploit the unique isotopic labeling pattern that signals the presence of a functional purinosome to survey human cell lines to ascertain the generality of this metabolon at endogenous levels. By extending our chemical imaging capabilities, we will show that the cellular “hot spots” are loci of the pathway enzymes and their respective metabolites. The determination of the actual number of enzyme copies within a purinosome will be undertaken with a novel application of STORM. The actual organization of the metabolon will be probed by a combination of intracellular photo- and enzyme- specific crosslinking. We will resort to APEX to validate the identity of nearest neighbors within the purinosome and to discover other adjacent proteins that emanate from nearby structures such as the mitochondrial membrane. Through a collaborative effort, cell lines that use a functioning purinosome for purine synthesis will be examined in cells and in xenografts to ascertain the metabolic requirements and the advantage of the metabolon for cellular proliferation. The proposed studies have important ramifications for characterizing the importance of the purinosome to human cell viability.