【摘要】 内质网-质膜连接是真核细胞一种新型的膜连接体系,介导内质网与质膜之间紧密的物质与信息交换.在条件致病真菌白念珠菌(Candida albicans)中,尚未明确内质网-质膜连接蛋白的种类与功能.本研究通过荧光定位观察发现, Ist2能够与白念珠菌内质网-质膜连接共定位,其缺失导致内质网-质膜连接程度明显减弱,表明该蛋白是白念珠菌一种关键的内质网-质膜连接蛋白.通过生长曲线测定、非折叠蛋白应答(unfolded protein response,UPR)报告体系荧光检测以及胞质钙含量测定发现,在衣霉素(tunicamycin, TN)造成的内质网压力条件下, IST2缺失菌株ist2Δ/Δ生长量明显增加, UPR报告基因PRB1的表达量下降,胞质钙含量降低.进一步采用钙离子螯合剂乙二醇双(2-氨基乙基醚)四乙酸(EGTA)与TN共处理发现, EGTA能够消除ist2Δ/Δ与野生型菌株之间内质网压力耐受性的差异.因此, Ist2作为白念珠菌的内质网-质膜连接蛋白,介导内质网压力条件下胞质钙浓度的增加,造成细胞内质网压力耐受性降低,是白念珠菌内质网压力应答的负调控因子.本研究丰富了对真菌内质网-质膜连接结构与功能的认识,为新型抗真菌药物靶点的开发提供了重要依据.更多还原

【Abstract】 The endoplasmic reticulum-plasma membrane contact（ER-PM contact） is a novel membrane contact system in eukaryotic cells that mediates the close exchange of materials and messengers between ER and PM. However, the types and function of the ER-PM tethering proteins of the opportunistic pathogenic fungus Candida albicans remain poorly understood. In the present study, we found that Ist2 co-localizes with the ER-PM contact sites of C. albicans and that its deletion causes a marked decrease in the ER-PM contact sites as revealed by fluorescence observation. This indicates that Ist2 is a key ER-PM tethering protein of C. albicans. The measurement of growth curves, fluorescence detection of the unfolded protein response（UPR） reporting system, and the measurement of the cytosolic Ca²⁺level revealed that the IST2 deletion strain ist2Δ/Δ considerably increased in biomass, the UPR reporting gene PRB1 expression was attenuated, and the cytoplasmic Ca²⁺levels under ER stress caused by tunicamycin decreased. In addition, the Ca²⁺chelating agent EGTA could eliminate the difference between ist2Δ/Δ and WT in ER stress tolerance. Therefore,Ist2 is an important ER-PM tethering protein in C. albicans and a negative regulator of ER stress responses, which mediates an increase in cytosolic Ca²⁺level under ER stress, resulting in attenuated ER stress tolerance. The results of the present study facilitate our understanding of the structure and function of the fungal ER-PM contact and provide an important basis for the development of novel antifungal targets.更多还原