Papouskova Klara, Jiang Linghuo, Sychrova Hana
Department of Membrane Transport, Institute of Physiology Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
The National Engineering Laboratory for Cereal Fermentation Technology, School of Biotechnology, Jiangnan University, Wuxi 214122, China The State Key Laboratory of Food Science and Technology, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
FEMS Yeast Res. 2015 Mar;15(2). doi: 10.1093/femsyr/fov007. Epub 2015 Feb 16.
Endosomal sorting complexes required for transport (ESCRTs) are involved in the formation of multivesicular bodies and sorting of targeted proteins to the yeast vacuole. The deletion of seven genes encoding components of the ESCRT machinery render Saccharomyces cerevisiae cells sensitive to high extracellular CaCl2 concentrations as well as to low pH in media. In this work, we focused on intracellular pH (pHin) homeostasis of these mutants. None of the studied ESCRT mutants exhibited an altered pHin level compared to the wild type under standard growth conditions. Nevertheless, 60 min of CaCl2 treatment resulted in a more significant drop in pHin levels in these mutants than in the wild type, suggesting that pHin homeostasis is affected in ESCRT mutants upon the addition of calcium. Similarly, CaCl2 treatment caused a bigger pHin decrease in cells lacking the vacuolar Ca(2+)/H(+) antiporter Vcx1 which indicates a role for this protein in the maintenance of proper pHin homeostasis when cells need to cope with a high CaCl2 concentration in media. Importantly, ESCRT gene deletions in the vcx1Δ strain did not result in an increase in the CaCl2-invoked drop in the pHin levels of cells, which demonstrates a genetic interaction between VCX1 and studied ESCRT genes.
转运所需的内体分选复合体（ESCRTs）参与多囊泡体的形成以及将靶向蛋白分选至酵母液泡。编码ESCRT机制组分的七个基因的缺失使酿酒酵母细胞对高细胞外氯化钙浓度以及培养基中的低pH敏感。在这项工作中，我们聚焦于这些突变体的细胞内pH（pHin）稳态。在标准生长条件下，与野生型相比，所研究的ESCRT突变体均未表现出pHin水平的改变。然而，氯化钙处理60分钟后，这些突变体的pHin水平下降幅度比野生型更大，这表明添加钙后ESCRT突变体的pHin稳态受到影响。同样，氯化钙处理导致缺乏液泡Ca(2+)/H(+)反向转运蛋白Vcx1的细胞中pHin下降幅度更大，这表明当细胞需要应对培养基中高浓度氯化钙时，该蛋白在维持适当的pHin稳态中发挥作用。重要的是，vcx1Δ菌株中ESCRT基因的缺失并未导致细胞中氯化钙引起的pHin水平下降增加，这证明了VCX1与所研究的ESCRT基因之间存在遗传相互作用。
