Purinosome formation as a function of the cell cycle

Abstract

The de novo purine biosynthetic pathway relies on six enzymes to catalyze the conversion of phosphoribosylpyrophosphate to inosine 5′-monophosphate. Under purine-depleted conditions, these enzymes form a multienzyme complex known as the purinosome. Previous studies have revealed the spatial organization and importance of the purinosome within mammalian cancer cells. In this study, time-lapse fluorescence microscopy was used to investigate the cell cycle dependency on purinosome formation in two cell models. Results in HeLa cells under purine-depleted conditions demonstrated a significantly higher number of cells with purinosomes in the G 1 phase, which was further confirmed by cell synchronization. HGPRT-deficient fibroblast cells also exhibited the greatest purinosome formation in the G 1 phase; however, elevated levels of purinosomes were also observed in the S and G 2 /M phases. The observed variation in cell cycle-dependent purinosome formation between the two cell models tested can be attributed to differences in purine biosynthetic mechanisms. Our results demonstrate that purinosome formation is closely related to the cell cycle.

A New View into the Regulation of Purine Metabolism: The Purinosome

“…Immunofluorescence has demonstrated purinosome formation on the endogenous level and provided evidence that the observed compartmentalization is not a consequence of overexpression due to transient transfection [6, 34-36]. Additionally, particle characterization in transient transfected models demonstrated that these enzyme clusters are distinct in size and cell density from processing bodies (P-bodies), stress granules, and aggresomes (Box 2) [37]. Using G3BP as a stress granule marker and gp250 and gp170* as aggresome markers, the purinosome enzyme cluster did not colocalize [38].…”

“…Using G3BP as a stress granule marker and gp250 and gp170* as aggresome markers, the purinosome enzyme cluster did not colocalize [38]. Finally, Western blot analysis found the levels of all six enzymes remained unchanged as the composition of the culturing media was switched [37]. Together, these conclusions further define the purinosome as a unique cellular body and not an artifact of transient transfection.…”

“…In addition, a 3-fold increase in the intracellular PRPP concentration accompanied this same cell cycle stage transition suggesting that the increased flux through the pathway is due in part to the increased concentrations of the pathway’s input substrate. Using time-lapse fluorescence microscopy, the percentage of purinosome-positive HeLa cells was found to be highest in G 1 -phase with a 3.8-fold increase in the purinosome containing cells arrested in the G 1 -phase by dibutyryl-cAMP [37]. As HeLa cells then progress through S-phase onto G 2 /M-phases, dramatic decreases in the purinosome content among the cell population were observed.…”

“…Under cellular conditions that promote purinosome formation, a 50% enhancement of metabolic flux was observed leading to a 3-fold increase in IMP formation compared to normal growth conditions [36]. Since the cells in these studies were asynchronous, we anticipate significant increases in the pathway flux during the G 1 phase of the cell cycle, where the number of purinosomes in a cell is highest [37]. Moreover, fibroblasts with loss-of-function mutations in HPRT1 , impeding IMP production by salvage and solely relying on the de novo pathway, showed a 25% increase in purinosome formation (Table 1) [55].…”

Detecting Purinosome Metabolon Formation with Fluorescence Microscopy

“…The spatial organization of these enzymes in cells is referred to as a purinosome and has been the subject of recent reviews [9, 10]. Purinosome assembly has shown to be a reversible phenomenon whose phenotype is largely predominant when cellular conditions result in a high purine demand, such as in the G 1 -phase of the cell cycle [6, 11]. Cellular conditions favoring purinosome formation were also shown to enhance the metabolic flux of the de novo purine biosynthetic pathway suggesting that the two observations are connected—a generalized hypothesis surrounding metabolon formation in cells [12].…”