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.

Francisca Randez‐Gil,Francisco Estruch,Jose A Prieto,Isabel E Sánchez‐Adriá

doi:10.1111/1751-7915.13513

Abstract

SummaryThe 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.

Highlights

Energy-generating metabolic pathways have a great influence on cell physiology, and understanding the mechanisms of their regulation is of major interest
Protein extracts from wild-type, tdh1, tdh2 and tdh1 tdh2 strains containing a chromosomal copy of GFP-tagged TDH3 were resolved by SDS–PAGE and visualized by Western blot using an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody
We observed a weaker signal in the tdh2 mutant samples, a result that is consistent with the low expression of TDH1 in cells growing at the exponential phase, as previously reported (McAlister and Holland, 1985)

Summary

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been traditionally considered a housekeeping protein involved in energy generation. 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. Like other GAPDHs, Tdh exists predominantly as a tetramer, dimeric and monomeric forms have been isolated. Mechanisms of Tdh regulation may imply changes in its oligomeric state or be based in its ability to interact with Tdh and/or Tdh to form hybrid complexes. No direct evidence of the existence of these interactions has been provided and the exact function of Tdh is unknown. Tdh tdh mutant cells show increased sensitivity to aureobasidin A, an inhibitor of the inositolphosphoryl ceramide synthase, while cells lacking Tdh showed improved tolerance. Engineering Tdh activity could be exploited to alter the SLs status with consequences in different aspects of yeast biotechnology

Introduction

Results and discussion

Concluding remarks

Experimental procedures