Enzymatic analysis of yeast cell wall-resident GAPDH and its secretion

Abstract

AbstractIn yeast, many proteins are found both in the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish non-conventional secretion from cellular leakage. We therefore monitored extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for non-conventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide crosslinks. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30-60-minute lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level one hour after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells.ImportanceEukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi, and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells, while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme, and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate non-conventional secretion more generally.