Characterization of the Saccharomyces cerevisiae sec6-41 mutation and tools to create S-cerevisiae strains containing the sec6-4 allele
Characterization of the Saccharomyces cerevisiae sec6-41 mutation and tools to create S-cerevisiae strains containing the sec6-4 allele
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DOI：
10.1016/j.gene.2005.07.014
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发表时间：
2005-11-21
期刊：
影响因子：
3.5
通讯作者：
Cannon, RD
中科院分区：
文献类型：
--
作者：
Lamping, E;Tanabe, K;Cannon, RD
真核细胞高度保守的胞囊复合体允许晚期分泌囊泡与质膜极化运输和融合。在酿酒酵母中，胞囊复合物的Sec6p组分对细胞生长至关重要。酿酒酵母SEC6基因的SEC6 -4温度敏感突变导致突变细胞在37℃的限制性温度下在细胞质中积累了大量成熟的晚期高尔基分泌囊泡，这些囊泡易于分离，内向外且密封紧密，含有成熟的翻译后修饰的质膜和分泌蛋白，为研究质膜蛋白功能提供了有价值的工具。本研究表明SEC6编码区的单点突变L633P决定了SEC6 -4表型。我们对野生型Sec6p和突变型Sec6-4p蛋白（c端标记为绿色荧光蛋白yEGfp3p）在存在或不存在异源过表达的白色念珠菌质膜atp结合盒（ABC）转运体CaCdr1p （c端标记为红色荧光蛋白mRfp1p）的情况下进行定位。与野生型Sec6p一样，Sec6-4p蛋白定位于芽和隔，在23℃的允许温度下，sec6-4突变体细胞的生长速度与野生型对照细胞相同。在37℃的限制温度下，Sec6-4p错位，细胞内积聚了异质囊泡，而在允许温度下，sec6-4细胞也积聚了同质的分泌囊泡。(c) 2005 Elsevier b.v.版权所有
The highly conserved exocyst complex of eukaryotic cells allows the polarized transport and fusion of late secretory vesicles with the plasma membrane. In Saccharomyces cerevisiae the Sec6p component of the exocyst complex is essential for cell growth. The sec6-4 temperature-sensitive mutation of the S. cerevisiae SEC6 gene leads to the accumulation of large amounts of mature late post-Golgi secretory vesicles in the cytosol of mutant cells at the restrictive temperature of 37 C. These readily isolated, inside-out and tightly sealed vesicles contain mature post-translationally modified plasma membrane and secretory proteins and provide a valuable tool for the study of plasma membrane protein function. This study shows that the single point mutation L633P in the SEC6 coding region defines the sec6-4 phenotype. We followed the localization of the wild type Sec6p and the mutant Sec6-4p proteins (C-terminally tagged with the green fluorescent protein yEGfp3p) in the presence or absence of heterologously over-expressed Candida albicans plasma membrane ATP-binding cassette (ABC) transporter CaCdr1p (C-terminally tagged with the red fluorescent protein mRfp1p). The Sec6-4p protein localized to buds and septa, like wild type Sec6p, at the permissive temperature of 23 C and the sec6-4 mutant cells grew at the same rate as the wild type control cells. Sec6-4p was mislocalized at the restrictive temperature of 37 C and heterogenous vesicles accumulated in cells but sec6-4 cells also accumulated homogenous secretory vesicles at the permissive temperature. (c) 2005 Elsevier B.V All rights reserved.