酿酒酵母全基因组SNARE蛋白的亚细胞定位研究  

A Subcellular Localization Survey for All SNARE Proteins in Saccharomyces cerevisiae

作　　者：张正坦 朱婧 谢志平 ZHANG Zheng-tan;ZHU Jing;XIE Zhi-ping(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室

出　　处：《中国生物工程杂志》2019年第10期44-57,共14页China Biotechnology

基　　金：上海市自然科学基金探索类项目(18ZR1420400);上海市教育委员会科研创新计划(2017-01-07-00-02-E00035)资助项目

摘　　要：在真核细胞中,内质网、高尔基体、质膜等膜结构间的蛋白质运输主要通过囊泡出芽和融合实现。SNARE蛋白家族在介导囊泡与目的膜结构融合过程中发挥关键作用。在模式生物酿酒酵母中,对全基因组SNARE蛋白的系统研究仍有不足。此研究构建了一套用于标记酿酒酵母基因组全部24种SNARE的工具质粒。该系列质粒既能呈现出良好的定位特征,又避免了过度表达造成的定位异常。通过与细胞器标记共定位验证了SNARE蛋白的亚细胞定位。结果发现3种SNARE的定位与之前报道不符:Bos1定位于早高尔基体,Snc1和Bet1定位于晚高尔基体/早内体。另外,Sec9定位于芽尖和芽颈,这是首次观察到Sec9在活酵母细胞中的定位。这项工作首次全面的检验了酵母SNARE家族蛋白的亚细胞定位,为后续SNARE蛋白功能研究提供了新线索,并为相关研究提供了一套工具质粒。In eukaryotic cells,protein transport between membrane structures such as endoplasmic reticulum,Golgi,and plasma membrane is mainly achieved via vesicle budding and fusion.The SNARE protein family plays a key role in mediating the fusion of vesicles with the target membrane structure.In the model organism Saccharomyces cerevisiae,systematic studies of SNARE proteins in the whole genome are still lacking.A set of plasmids for the labeling of all 24 SNAREs in S.cerevisiae with GFP are constructed.Most of the plasmids employ endogenous promoters for expression,with only a few mildly overexpressed,thus avoiding potential mislocalization caused by high overexpression.The subcellular localization of each SNARE is verified by co-localization with organelle markers.Results indicate that the localization of three SNAREs differs from those in existing literature:Bos1 localizes to early Golgi;and Snc1 and Bet1 localize to the late Golgi/early endosomes.In addition,Sec9 is detected at the bud tip and septum.This is the first time that the localization of Sec9 in vegetative cells has been observed.It is also the first comprehensive experimental evaluation of yeast SNARE subcellular localization.Furthermore,the constructed plasmids constitute a convenient tool set for future yeast cell biology studies.

关 键 词：SNARE 亚细胞定位 酿酒酵母 共定位 

分 类 号：Q816[生物学—生物工程]