posted on 2016-04-29, 00:00authored byDanielle
L. Schmitt, Yun-ju Cheng, Junyong Park, Songon An
Dynamic
partitioning of de novo purine biosynthetic
enzymes into multienzyme compartments, purinosomes, has been associated
with increased flux of de novo purine biosynthesis
in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells.
We have investigated the functional role of AMP-activated protein
kinase (AMPK) in the regulation of de novo purine
biosynthesis because of its regulatory action on lipid and carbohydrate
biosynthetic pathways. Using pharmacological AMPK activators, we have
monitored subcellular localizations of six pathway enzymes tagged
with green fluorescent proteins under time-lapse fluorescence single-cell
microscopy. We revealed that only one out of six pathway enzymes,
formylglycinamidine ribonucleotide synthase (FGAMS), formed spatially
distinct cytoplasmic granules after treatment with AMPK activators,
indicating the formation of single-enzyme self-assemblies. In addition,
subsequent biophysical studies using fluorescence recovery after photobleaching
showed that the diffusion kinetics of FGAMS were slower when it localized
inside the self-assemblies than within the purinosomes. Importantly,
high-performance liquid chromatographic studies revealed that the
formation of AMPK-promoted FGAMS self-assembly caused the reduction
of purine metabolites in HeLa cells, indicating the downregulation
of de novo purine biosynthesis. Collectively, we
demonstrate here that the spatial sequestration of FGAMS by AMPK is
a mechanism by which de novo purine biosynthesis
is downregulated in human cells.