Purinosome formation as a function of the cell cycle

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 G₁ phase, which was further confirmed by cell synchronization. HGPRT-deficient fibroblast cells also exhibited the greatest purinosome formation in the G₁ phase; however, elevated levels of purinosomes were also observed in the S and G₂/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.Enzymes have been shown to form clusters in a cell to regulate metabolic processes (1–3). More recently, the concept of multienzyme complexes has been expanded to include mesoscale protein assemblies that appear to be substantially larger than a single protein (4). Depending on the metabolic or developmental state of the cells, these types of protein clusters range from transiently associated to very rigid and well defined (4). Examples of such enzyme clusters include the EF-Tu cytoskeleton, RNA degradosome, CTP synthase, carboxysomes, and nucleolus (5–9).In humans, purine nucleotides are synthesized by two different mechanisms. The first mechanism, de novo purine biosynthesis, converts phosphoribosylpyrophosphate (PRPP) to inosine 5′-phosphate (IMP) in 10 highly conserved steps catalyzed by six enzymes. These six enzymes include one trifunctional enzyme (TrifGART: GARS, GART, and AIRS domains), two bifunctional enzymes (PAICS: CAIRS and SAICARS domains; ATIC: AICART and IMPCH domains), and three monofunctional enzymes (PPAT, FGAMS, and ASL). ASL is also necessary for the conversion of IMP to AMP and may be classified as bifunctional (10). The second mechanism uses nucleotide salvage pathways to either phosphorylate a nucleoside (e.g., thymidine kinase) or add a purine base to ribose 5′-phosphate to regenerate the respective monophosphate. For example, hypoxanthine/guanine phosphoribosyl transferase (HGPRT) catalyzes the conversion of hypoxanthine to IMP and the conversion of guanine to guanosine 5′-phosphate (GMP). The de novo pathway is more energy-intensive, with the synthesis of 1 mole of IMP requiring 5 moles of ATP. Therefore, the salvage pathway is the preferred pathway for purine biosynthesis. Cells deficient in HGPRT, such as those that exhibit a Lesch–Nyhan disease (LND) phenotype, rely primarily on the de novo purine biosynthetic pathway to generate purine nucleotides (11–15).Recently, enzymes in the de novo purine biosynthetic pathway were shown to organize and reversibly assemble into punctate cellular bodies known as “purinosomes” under purine-depleted conditions (16). Further investigations into the organization of the purinosome showed that several of the enzymes form a core structure (PPAT, TrifGART, and FGAMS), whereas others appear to interact peripherally (PAICS, ASL, and ATIC) (17). The presence of this protein assembly and its putative function(s) clearly suggest that the spatial organization of pathway enzymes into purinosomes within a cell play an important role in meeting the cellular demand for purines (18). Although many studies have implied up-regulation of the de novo purine biosynthetic pathway during cell cycle progression (14, 19–21), here we used time-lapse microscopy to determine whether a correlation exists between the cell cycle stage and the number of cells with purinosomes or the cells’ purinosome content. Two different cell types, HeLa and LND cells, were used, with the latter deficient in purine salvage, to assess the effect of increased demand on the de novo pathway for purine biosynthesis and the attendant consequences for purinosome assembly.