Detecting Purinosome Metabolon Formation with Fluorescence Microscopy.

A long-standing hypothesis in the de novo purine biosynthetic pathway is that there must be highly coordinated processes to allow for enhanced metabolic flux when a cell demands purines. One mechanism by which the pathway meets its cellular demand is through the spatial organization of pathway enzymes into multienzyme complexes called purinosomes. Cellular conditions known to impact the activity of enzymes in the pathway or overall pathway flux have been reflected in a change in the number of purinosome-positive cells or the density of purinosomes in a given cell. The following general protocols outline the steps needed for purinosome detection through transient expression of fluorescent protein chimeras or through immunofluorescence in purine-depleted HeLa cells using confocal laser scanning microscopy. These protocols define a purinosome as a colocalization of FGAMS with one additional pathway enzyme, such as PPAT or GART, and provide insights into the proper identification of a purinosome from other reported cellular bodies.