Purines are synthesized de novo in 10 chemical steps that are catalyzed by six enzymes in eukaryotes. Studies in vitro have provided little evidence of anticipated protein-protein interactions that would enable substrate channeling and regulation of the metabolic flux. We applied fluorescence microscopy to HeLa cells and discovered that all six enzymes colocalize to form clusters in the cellular cytoplasm. The association and dissociation of these enzyme clusters can be regulated dynamically, by either changing the purine levels of or adding exogenous agents to the culture media. Collectively, the data provide strong evidence for the formation of a multi-enzyme complex, the "purinosome," to carry out de novo purine biosynthesis in cells.

Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells.

ProlylPro . This function appears to be redundant with that of its corresponding ProRS, which contains a canonical bacterial editing domain. In the present study, we test the specificity of editing by H. influenzae YbaK and show that it efficiently edits Cys-tRNA Pro and that a conserved Lys residue is essential for this activity. These findings represent the first example of an editing domain paralog possessing altered specificity and suggest that similar autonomous editing domains could act upon different mischarged tRNAs thus providing cells with enhanced proofreading potential. This work also suggests a novel mechanism of editing wherein a third sieve is used to clear CystRNA Pro in at least some organisms.

The organization of metabolic multienzyme complexes has been hypothesized to benefit metabolic processes and provide a coordinated way for the cell to regulate metabolism. Historically, their existence has been supported by various in vitro techniques. However, it is only recently that the existence of metabolic complexes inside living cells has come to light to corroborate this long-standing hypothesis. Indeed, subcellular compartmentalization of metabolic enzymes appears to be widespread and highly regulated. On the other hand, it is still challenging to demonstrate the functional significance of these enzyme complexes in the context of the cellular milieu. In this review, we discuss the current understanding of metabolic enzyme complexes by primarily focusing on central carbon metabolism and closely associated metabolic pathways in a variety of organisms, as well as their regulation and functional contributions to cells.

Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cellbased assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells.metabolism | protein complex | purine biosynthesis