The formation of hybrid complexes between isoenzymes of glyceraldehyde‐3‐phosphate dehydrogenase regulates its aggregation state, the glycolytic activity and sphingolipid status in Saccharomyces cerevisiae

Abstract

Summary The glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has been traditionally considered a housekeeping protein involved in energy generation. However, evidence indicates that GAPDHs from different origins are tightly regulated and that this regulation may be on the basis of glycolysis‐related and glycolysis‐unrelated functions. In Saccharomyces cerevisiae, Tdh3 is the main GAPDH, although two other isoenzymes encoded by TDH1 and TDH2 have been identified. Like other GAPDHs, Tdh3 exists predominantly as a tetramer, although dimeric and monomeric forms have also been isolated. Mechanisms of Tdh3 regulation may thus imply changes in its oligomeric state or be based in its ability to interact with Tdh1 and/or Tdh2 to form hybrid complexes. However, no direct evidence of the existence of these interactions has been provided and the exact function of Tdh1,2 is unknown. Here, we show that Tdh1,2 immunopurified with a GFP‐tagged version of Tdh3 and that lack of this interaction stimulates the Tdh3’s aggregation. Furthermore, we found that the combined knockout of TDH1 and TDH2 promotes the loss of cell’s viability and increases the growing rate, glucose consumption and CO2 production, suggesting a higher glycolytic flux in the mutant cells. Consistent with this, the tdh3 strain, which displays impaired in vitro GAPDH activity, exhibited the opposite phenotypes. Quite remarkably, tdh1 tdh2 mutant cells show increased sensitivity to aureobasidin A, an inhibitor of the inositolphosphoryl ceramide synthase, while cells lacking Tdh3 showed improved tolerance. The results are in agreement with a link between glycolysis and sphingolipid (SLs) metabolism. Engineering Tdh activity could be thus exploited to alter the SLs status with consequences in different aspects of yeast biotechnology.

Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

“…Glucose-6-phosphate and ATP can therefore be generated from glycerol, which represses the glyoxylate cycle and the consumption of ethanol in K. lactis [ 47 , 48 ]. The GAPDH isoforms in S. cerevisiae were shown to form heteromeric complexes, which may well occur with the K. lactis enzymes, too [ 42 ]. Concerning the importance of the different isoforms in vivo, KlTdh2 seems to be more reminiscent of ScTdh3, as a lack of the latter also displayed a slight growth defect on glucose media [ 42 ].…”

“…The GAPDH isoforms in S. cerevisiae were shown to form heteromeric complexes, which may well occur with the K. lactis enzymes, too [ 42 ]. Concerning the importance of the different isoforms in vivo, KlTdh2 seems to be more reminiscent of ScTdh3, as a lack of the latter also displayed a slight growth defect on glucose media [ 42 ]. The authors also proposed another role for GAPDH in yeast sphingolipid metabolism, whose conservation in K. lactis remains to be determined.…”

“…In S. cerevisiae , all three isoforms were detected in the cell wall, suggesting an extracellular function [ 41 ]. Moreover, the three isoforms were identified as the source of antifungal peptides claimed to ensure the predominance of S. cerevisiae in the later stages of wine fermentations [ 42 ]. GAPDH from the opportunistic pathogen Candida albicans was found in the cell wall as an immunoactive protein, antibodies which can prevent systemic infections caused by this yeast [ 43 ].…”

Genome-wide base editor screen identifies regulators of protein abundance in yeast

“…In S. cerevisiae, Tdh3 is thought to be the main GAPDH enzyme in glycolysis and is one of the most highly expressed proteins 14 . None of the three GAPDH genes are essential for cell viability, but a functional copy of either TDH2 or TDH3 is required; in contrast, cells with a TDH1 deletion, either alone or in combination with a TDH2 or a TDH3 deletion, are viable [40][41][42] . The three isoenzymes were shown to selectively change abundance in response to available carbon sources and other environmental perturbations [43][44][45][46] .…”

Glyceraldehyde-3-phosphate Dehydrogenase Is a Multifaceted Therapeutic Target

“…In addition, the activities of GAPDH may be regulated by redox reactions, for example S-thiolation, which appears to serve an adaptive function during exposure to an oxidative stress [6]. GAPDH is capable of functioning in the cell both in the enzymatically active, tetrameric form necessary for glycolysis, and in the dimeric or monomeric forms [7,8]. Moreover, the cellular localization of GAPDH is not limited to the cytoplasm, the protein is found in the nucleus and other intracellular organelles [9], including plasma membrane [10].…”