Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
mSphere. 2021 Dec 22;6(6):e0092921. doi: 10.1128/msphere.00929-21. Epub 2021 Dec 15.
The heterotrimeric protein kinase SNF1 is a key regulator of metabolic adaptation in the pathogenic yeast Candida albicans, and mutants with a defective SNF1 complex cannot grow on carbon sources other than glucose. We identified a novel type of suppressor mutation in the β-subunit Kis1 that rescued the growth defects of cells lacking the regulatory γ-subunit Snf4 of the SNF1 complex. Unlike wild-type Kis1, the mutated Kis1 could bind to the catalytic α-subunit Snf1 in the absence of Snf4. Binding of Kis1 did not enhance phosphorylation of Snf1 by the upstream activating kinase Sak1, which is impaired in Δ mutants. Nevertheless, the mutated Kis1 reestablished SNF1-dependent gene expression, confirming that SNF1 functionality was restored. The repressor proteins Mig1 and Mig2 were phosphorylated even in the absence of Snf1, but their phosphorylation patterns were altered, indicating that SNF1 regulates Mig1 and Mig2 activity indirectly. In contrast to wild-type cells, mutants lacking Snf4 were unable to reduce the amounts of Mig1 and Mig2 when grown on alternative carbon sources, and this deficiency was also remediated by the mutated Kis1. These results provide novel insights into the regulation of SNF1 and the repressors Mig1 and Mig2 in the metabolic adaptation of C. albicans. The highly conserved protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans, but it is not clear how it regulates its downstream targets in this fungus. We show that the repressor proteins Mig1 and Mig2 are phosphorylated also in cells lacking the catalytic α-subunit Snf1 of the SNF1 complex, but the amounts of both proteins were reduced in wild-type cells when glucose was replaced by alternative carbon sources, pointing to an indirect mechanism of regulation. Mutants lacking the regulatory γ-subunit Snf4 of the SNF1 complex, which cannot grow on alternative carbon sources, were unable to downregulate Mig1 and Mig2 levels. We identified a novel type of suppressor mutation, an amino acid substitution in the β-subunit Kis1, which enabled Kis1 to bind to Snf1 in the absence of Snf4, thereby restoring Mig1 and Mig2 downregulation, SNF1-dependent gene expression, and growth on alternative carbon sources. These results provide new insights into the SNF1 signaling pathway in C. albicans.
异三聚体蛋白激酶 SNF1 是致病性酵母白色念珠菌代谢适应的关键调节因子，而 SNF1 复合物缺陷突变体不能在除葡萄糖以外的碳源上生长。我们在 β 亚基 Kis1 中发现了一种新型的抑制突变，该突变挽救了 SNF1 复合物缺失调节 γ 亚基 Snf4 的细胞的生长缺陷。与野生型 Kis1 不同，突变型 Kis1 可以在没有 Snf4 的情况下与催化α亚基 Snf1 结合。Kis1 的结合并没有增强上游激活激酶 Sak1 对 Snf1 的磷酸化，而这种磷酸化在 Δ 突变体中受到损害。然而，突变型 Kis1 重新建立了 SNF1 依赖性基因表达，证实了 SNF1 功能的恢复。即使在没有 Snf1 的情况下，阻遏蛋白 Mig1 和 Mig2 也被磷酸化，但它们的磷酸化模式发生了改变，表明 SNF1 间接调节 Mig1 和 Mig2 的活性。与野生型细胞不同，当在替代碳源上生长时，缺失 Snf4 的突变体不能减少 Mig1 和 Mig2 的数量，而突变型 Kis1 也可以弥补这一缺陷。这些结果为白色念珠菌代谢适应中 SNF1 和阻遏蛋白 Mig1 和 Mig2 的调节提供了新的见解。高度保守的蛋白激酶 SNF1 在致病性酵母白色念珠菌的代谢适应中起着关键作用，但目前尚不清楚它如何在这种真菌中调节其下游靶标。我们表明，即使在缺失 SNF1 复合物的催化α亚基 Snf1 的情况下，阻遏蛋白 Mig1 和 Mig2 也被磷酸化，但在葡萄糖被替代碳源取代时，野生型细胞中这两种蛋白的数量都会减少，这表明存在一种间接的调节机制。不能在替代碳源上生长的 SNF1 复合物缺失调节γ亚基 Snf4 的突变体，无法下调 Mig1 和 Mig2 水平。我们发现了一种新型的抑制突变，即 Kis1 的β亚基中的一个氨基酸取代，该突变使 Kis1 能够在没有 Snf4 的情况下与 Snf1 结合，从而恢复 Mig1 和 Mig2 的下调、SNF1 依赖性基因表达以及在替代碳源上的生长。这些结果为白色念珠菌中的 SNF1 信号通路提供了新的见解。
