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

Abstract

The authors show that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Saccharomyces cerevisiae, previously thought to be restricted to the cell interior, is also present in the cell wall. GAPDH activity, proportional to cell number and time of incubation, was detected in intact wild-type yeast cells. Intact cells of yeast strains containing insertion mutations in each of the three structural TDH genes (tdh1, tdh2 and tdh3) and double mutants (tdh1 tdh2 and tdh1 tdh3) also displayed a cell-wall-associated GAPDH activity, in the range of parental wild-type cells, although with significant differences among strains. A cell wall location of GAPDH was further confirmed in wild-type and tdh mutants by indirect immunofluorescence and flow cytometry analysis with a polyclonal antibody against S. cerevisiae GAPDH. By immunoelectron microscopy, the GAPDH protein was detected 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 cytosol showed that Tdh2 and Tdh3 polypeptides are present in the cell wall, as well as in the cytosol, of exponentially growing cells. Tdh1 is only detected 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 which retain their catalytic activity.

Conserved Processes and Lineage-Specific Proteins in Fungal Cell Wall Evolution

“…Their primary locations are intracellular. Although their role as cell wall proteins is less well known and somewhat controversial, it is well documented in two ascomycetous yeasts, S. cerevisiae and C. albicans (14,30,40,41,46). Given their controversial or auxiliary role in cell walls and their dual roles as wall resident and key metabolic activities, these classes could be excluded from our analysis ("chaperones" and "metabolism" in Fig.…”

“…Forty of the 187 proteins had homologs in other kingdoms. These 40 ORFs were in the functional groups most conserved in the fungi, including glycosylases, GPI synthetic proteins, aspartyl proteases, a group of related proteins important for plant pathogenesis, and two cytoplasmic groups with known or suspected cell wall involvement: chaperones and metabolic enzymes (14,30,40,41,46). Because these kinds of genes are present in many eukaryote kingdoms, they are likely to be ancestral to all eukaryotes, including fungi.…”

“…Only about 10% of the proteins are reported with intracellular localization, including paralogs of chaperones and glycolytic enzymes. Those two classes of proteins are primarily cytoplasmic, but some proteins in each class are also proposed to be localized in cell walls in S. cerevisiae and in C. albicans on the bases of analyses of isolated walls and/or immunoassays on intact cells (14,30,40,41,46).…”

Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraldehyde‐3‐phosphate dehydrogenase‐invertase fusion protein

“…GAPDHs are cell wall-associated proteins in both C. albicans and S. cerevisiae cells (Gil-Navarro et al, 1997;Delgado et al, 2001). These proteins lack any conventional signal peptide responsible for its targeting into the yeast secretory pathway; thus, alternative mechanisms for their secretion should be envisaged.…”

“…Besides, in Candida albicans an immunogenic and enzymatically active cell wall-associated GAPDH is found at the cell surface (Gil-Navarro et al, 1997), and C. albicans clinical strains express this surface antigen both in vitro and in infected tissues ; since C. albicans GAPDH is also a fibronectin-and laminin-binding protein, the surface GAPDH could be involved in the adhesion of fungal cells to host tissues, thus playing a role in its virulence . In addition, the GAPDH polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wallassociated proteins, although the precise role of these cell surface GAPDHs in S. cerevisiae physiology has yet to be determined (Delgado et al, 2001).…”