Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
PLoS Genet. 2023 Aug 10;19(8):e1010890. doi: 10.1371/journal.pgen.1010890. eCollection 2023 Aug.
Protein kinases are central components of almost all signaling pathways that control cellular activities. In the model organism Saccharomyces cerevisiae, the paralogous protein kinases Ypk1 and Ypk2, which control membrane lipid homeostasis, are essential for viability, and previous studies strongly indicated that this is also the case for their single ortholog Ypk1 in the pathogenic yeast Candida albicans. Here, using FLP-mediated inducible gene deletion, we reveal that C. albicans ypk1Δ mutants are viable but slow-growing, explaining prior failures to obtain null mutants. Phenotypic analyses of the mutants showed that the functions of Ypk1 in regulating sphingolipid biosynthesis and cell membrane lipid asymmetry are conserved, but the consequences of YPK1 deletion are milder than in S. cerevisiae. Mutational studies demonstrated that the highly conserved PDK1 phosphorylation site T548 in its activation loop is essential for Ypk1 function, whereas the TORC2 phosphorylation sites S687 and T705 at the C-terminus are important for Ypk1-dependent resistance to membrane stress. Unexpectedly, Pkh1, the single C. albicans orthologue of Pkh1/Pkh2, which mediate Ypk1 phosphorylation at the PDK1 site in S. cerevisiae, was not required for normal growth of C. albicans under nonstressed conditions, and Ypk1 phosphorylation at T548 was only slightly reduced in pkh1Δ mutants. We found that another protein kinase, Pkh3, whose ortholog in S. cerevisiae cannot substitute Pkh1/2, acts redundantly with Pkh1 to activate Ypk1 in C. albicans. No phenotypic effects were observed in cells lacking Pkh3 alone, but pkh1Δ pkh3Δ double mutants had a severe growth defect and Ypk1 phosphorylation at T548 was completely abolished. These results establish that Ypk1 is not essential for viability in C. albicans and that, despite its generally conserved function, the Ypk1 signaling pathway is rewired in this pathogenic yeast and includes a novel upstream kinase to activate Ypk1 by phosphorylation at the PDK1 site.
蛋白激酶是几乎所有控制细胞活动的信号通路的核心组成部分。在模式生物酿酒酵母中，同源蛋白激酶 Ypk1 和 Ypk2 控制着膜脂的动态平衡，对酵母的生存是必需的，之前的研究强烈表明，这也是它们在病原性酵母白色念珠菌中的单一同源物 Ypk1 的情况。在这里，我们使用 FLP 介导的诱导性基因缺失，揭示了白色念珠菌 ypk1Δ 突变体是有活力的，但生长缓慢，这解释了之前未能获得缺失突变体的原因。对突变体的表型分析表明，Ypk1 调节鞘脂生物合成和细胞膜脂质不对称性的功能是保守的，但 YPK1 缺失的后果比在酿酒酵母中要轻微。突变研究表明，其激活环中高度保守的 PDK1 磷酸化位点 T548 对于 Ypk1 的功能是必需的，而 C 端的 TORC2 磷酸化位点 S687 和 T705 对于 Ypk1 依赖的膜应激抗性是重要的。出乎意料的是，白色念珠菌中 Pkh1 的单一同源物 Pkh1/Pkh2 在酿酒酵母中介导 Ypk1 在 PDK1 位点的磷酸化，在非胁迫条件下，对于白色念珠菌的正常生长不是必需的，并且在 pkh1Δ 突变体中 Ypk1 在 T548 处的磷酸化仅略有减少。我们发现，另一种蛋白激酶 Pkh3 的同源物在酿酒酵母中不能替代 Pkh1/2，在白色念珠菌中与 Pkh1 一起发挥冗余作用来激活 Ypk1。单独缺失 Pkh3 的细胞没有观察到表型效应，但 pkh1Δ pkh3Δ 双突变体的生长缺陷严重，并且 Ypk1 在 T548 处的磷酸化完全被消除。这些结果表明，Ypk1 对于白色念珠菌的生存不是必需的，并且尽管其具有普遍保守的功能，但是 Ypk1 信号通路在这种病原性酵母中被重新布线，包括一种新的上游激酶，通过 PDK1 位点的磷酸化来激活 Ypk1。
