Abstract
In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional 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 100 μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. 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- to 60-min 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 1 h 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.IMPORTANCE Eukaryotic 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 nonconventional secretion more generally.
Highlights
In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and it can be difficult to distinguish nonconventional secretion from cellular leakage
We verified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is enzymatically active in the wall of S. cerevisiae strain BY4743 by resuspending cells in 1 mM NAD, 1 mM glyceraldehyde-3-phosphate, 100 mM dithiothreitol (DTT), and triethanolamine phosphate (TEA) buffer in a 200-ml reaction mixture, using methods similar to those of Delgado et al [1]
Yeast cells were pelleted by centrifugation, and NADH was measured as the A340
Summary
Many proteins are found in both the cytoplasmic and extracellular compartments, and it can be difficult to distinguish nonconventional secretion from cellular leakage. There were about 4 Â 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h 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. The cell walls consist of polysaccharides, including b1,3 glucans, b1,6 glucans, chitin, and a large number of proteins These cell wall-resident proteins cross-link the saccharides, act as adhesins, regulate metabolic activities, and perform other functions [24,25,26]. Most of these proteins are secreted through the conventional secretion signal-dependent pathway that processes the proteins through the endoplasmic reticulum (ER) and Golgi membrane [27, 28]. Temperature-sensitive S. cerevisiae secretory mutants were generated, and at nonpermissive temperatures they showed defects in invertase and acid phosphatase secretion [29]
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