Compartmentalization of Metabolic Pathways and Their Influence on Cellular Energetic

Metabolic enzymes have been hypothesized to compartmentalize in cells to facilitate in metabolic control. Indeed, there are several lines of evidence from across species which demonstrate the compartmentalization of single or multiple metabolic enzymes in cells. In human cells, enzymes in de novo purine biosynthesis or glycolysis and gluconeogenesis have the propensity to compartmentalize in the cytoplasm. The formation of these multi-enzyme compartments has been hypothesized to stabilize metabolic intermediates, facilitate in substrate channeling, and alter metabolic flux. Therefore, we propose that the compartmentalization of metabolic enzymes provides a means for the cell to regulate metabolism.;Here, we provide evidence of spatiotemporal regulatory mechanisms for de novo purine biosynthesis or glycolysis in human cancer cells. First, we reveal a novel downregulation mechanism of de novo purine biosynthesis. One of six enzymes involved in purine biosynthesis is sequestered upon pharmacological activation of AMP activated protein kinase (AMPK). This selective enzyme sequestration results in a measurable and significant decrease in purine nucleotide levels. Second, we find that phosphoinositide-dependent protein kinase 1 (PDK1) signaling pathways induce the clustering of the first three of six enzymes involved in purine biosynthesis. Using pharmacological inhibitors of Akt, PDK1, and their respective downstream effectors, we determine these enzymes co-cluster in an Akt-independent, PDK1-dependent manner. Third, we demonstrate the compartmentalization of a human glycolytic enzyme, liver-type phosphofructokinase I (PFKL), can be induced by small molecules. Using a quantitative high-content high-throughput screening assay, we find that inhibition of proteins involved in cell cycle progression results in the compartmentalization of PFKL in the cytoplasm. Collectively, our work provides a new insight into metabolic control in human cells, which may result in innovative advances in the treatment of metabolic disorders.