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.

中文翻译：

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3-磷酸​​甘油醛脱氢酶同工酶之间杂合复合物的形成调节其在酿酒酵母中的聚集状态、糖酵解活性和鞘脂状态。

糖酵解酶甘油醛-3-磷酸脱氢酶 (GAPDH) 传统上被认为是一种参与能量产生的管家蛋白。然而，有证据表明来自不同来源的 GAPDHs 受到严格调控，这种调控可能是基于糖酵解相关和糖酵解无关的功能。在酿酒酵母中，Tdh3 是主要的 GAPDH，尽管由TDH1和TDH2编码的另外两种同工酶已被识别。与其他 GAPDH 一样，Tdh3 主要以四聚体形式存在，尽管也已分离出二聚体和单体形式。因此，Tdh3 调控机制可能意味着其寡聚状态的变化或基于其与 Tdh1 和/或 Tdh2 相互作用形成混合复合物的能力。然而，没有提供这些相互作用存在的直接证据，并且 Tdh1,2 的确切功能是未知的。在这里，我们展示了用 GFP 标记的 Tdh3 版本免疫纯化的 Tdh1,2，并且缺乏这种相互作用会刺激 Tdh3 的聚集。此外，我们发现TDH1和TDH2的联合敲除促进了细胞活力的丧失，并增加了生长速率、葡萄糖消耗和CO ₂生产，表明突变细胞中的糖酵解通量更高。与此一致，tdh3菌株表现出体外GAPDH 活性受损，表现出相反的表型。非常显着的是，tdh1 tdh2突变细胞对肌醇磷酰神经酰胺合酶抑制剂 aureobasidin A 表现出更高的敏感性，而缺乏 Tdh3 的细胞表现出更高的耐受性。结果与糖酵解和鞘脂 (SLs) 代谢之间的联系一致。因此，可以利用工程 Tdh 活性来改变 SL 状态，从而在酵母生物技术的不同方面产生影响。