Data_Sheet_1_Decreased Vacuolar Ca2+ Storage and Disrupted Vesicle Trafficking Underlie Alpha-Synuclein-Induced Ca2+ Dysregulation in S. cerevisiae.docx

The yeast Saccharomyces cerevisiae is a powerful model to study the molecular mechanisms underlying α-synuclein (α-syn) cytotoxicity. This is due to the high degree of conservation of cellular processes with higher eukaryotes and the fact that yeast does not endogenously express α-synuclein. In this work, we focused specifically on the interplay between α-syn and intracellular Ca2+ homeostasis. Using temperature-sensitive SEC4 mutants and deletion strains for the vacuolar Ca2+ transporters Pmc1 and Vcx1, together with aequorin-based Ca2+ recordings, we show that overexpression of α-syn shifts the predominant temporal pattern of organellar Ca2+ release from a biphasic to a quasi-monophasic response. Fragmentation and vesiculation of vacuolar membranes in α-syn expressing cells can account for the faster release of vacuolar Ca2+. α-Syn further significantly reduced Ca2+ storage resulting in increased resting cytosolic Ca2+ levels. Overexpression of the vacuolar Ca2+ ATPase Pmc1 in wild-type cells prevented the α-syn-induced increase in resting Ca2+ and was able to restore growth. We propose that α-syn-induced disruptions in Ca2+ signaling might be an important step in initiating cell death.

酿酒酵母（Saccharomyces cerevisiae）是研究α-突触核蛋白（α-synuclein, α-syn）细胞毒性分子机制的经典模型。这是由于其细胞通路与高等真核生物具有高度保守性，且酵母内源不会表达α-突触核蛋白。本研究聚焦于α-syn与细胞内钙稳态之间的相互作用。通过使用温度敏感型SEC4突变体、液泡钙转运蛋白Pmc1与Vcx1的缺失菌株，结合基于水母素（aequorin）的钙信号记录实验，我们证实α-syn过表达会将细胞器钙释放的典型时序模式从双相反应转变为准单相反应。表达α-syn的细胞中出现的液泡膜碎片化与囊泡化现象，可解释液泡钙释放速率加快的原因。此外，α-syn还会显著降低细胞钙储存能力，导致静息胞质钙水平升高。在野生型细胞中过表达液泡钙ATP酶Pmc1，能够阻断α-syn诱导的静息钙水平升高，并恢复细胞生长。我们据此提出，α-syn引发的钙信号通路紊乱可能是启动细胞死亡的关键环节。