The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins

The authors show that the glycolytic enzyme glyceraldehyde-3-phosphate dehy drogenase (GAPDH) of Saccharomyces cerevisiae, previously thought to be res tricted to the cell interior, is also present in the cell wall. GAPDH activ ity, proportional to cell number and time of incubation, was detected in in tact wild-type yeast cells. Intact cells of yeast strains containing insert ion mutations in each of the three structural TDH genes (tdh1, tdh2 and tdh 3) and double mutants (tdh1 tdh2 and tdh1 tdh3) also displayed a cell-wall- associated GAPDH activity, in the range of parental wild-type cells, althou gh with significant differences among strains. A cell wall location of GAPD H was further confirmed in wild-type and tdh mutants by indirect immunofluo rescence and flow cytometry analysis with a polyclonal antibody against S c erevisiae GAPDH. By immunoelectron microscopy, the GAPDH protein was detect ed at the outer surface of the cell wall of wild-type cells, as well as in the cytoplasm. Western immunoblot analysis of cell wall extracts and cytoso l showed that Tdh2 and Tdh3 polypeptides are present in the cell wall, as w ell as in the cytosol. of exponentially growing cells. Tdh1 is only detecte d in stationary-phase cells, again in both cytosol and cell wall extracts. The results incorporate the GAPDH of S cerevisiae, encoded by TDH1-3, into the newly emerging family of multifunctional cell-wall-associated GAPDHs wh ich retain their catalytic activity.