Pga1 is an essential component of glycosylphosphatidylinositol-mannosyltransferase II of Saccharomyces cerevisiae
Pga1 is an essential component of glycosylphosphatidylinositol-mannosyltransferase II of Saccharomyces cerevisiae
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DOI：
10.1091/mbc.e07-03-0258
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发表时间：
2007-09-01
影响因子：
3.3
通讯作者：
Yoda, Koji
中科院分区：
文献类型：
--
作者：
Sato, Keisuke;Noda, Yoichi;Yoda, Koji
酿酒酵母（Saccharomycescerevisiae）必需基因YNL 158 w/PGA 1编码一种位于内质网（ER）的膜蛋白。我们通过易错聚合酶链反应突变技术构建了PGA 1的温度敏感等位基因，以探讨其生物学作用。脉冲追踪实验表明，pga 1（ts）突变体在限制性温度下积累了Gas 1蛋白的ER形式前体。蔗糖酶和羧肽酶Y的运输没有受到影响。TritonX-114相分离和[H-3]肌醇标记表明，糖基磷脂酰肌醇（GPI）锚定在pga 1（ts）突变体中是缺陷的，表明Pga 1参与GPI合成或其转移到靶蛋白。我们发现GPI 18，这是最近报道的编码GPI-MT II（GPI-MT II），作为一个高拷贝抑制剂的温度敏感性的pga 1（ts）。通过免疫荧光法在ER中检测到Gpi 18和Pga 1，并且它们从Triton X-100溶解的膜共沉淀。gpi 18（ts）和pga 1（ts）突变体在限制性温度下积累相同的GPI合成中间体。从这些结果，我们得出结论，Pga 1是一个额外的必要组成部分的酵母GPI-MT II。
The Saccharomyces cerevisiae essential gene YNL158w/PGA1 encodes an endoplasmic reticulum (ER)-localized membrane protein. We constructed temperature-sensitive alleles of PGA1 by error-prone polymerase chain reaction mutagenesis to explore its biological role. Pulse-chase experiments revealed that the pga1(ts) mutants accumulated the ER-form precursor of Gas1 protein at the restrictive temperature. Transport of invertase and carboxypeptidase Y were not affected. Triton X-114 phase separation and [H-3]inositol labeling indicated that the glycosylphosphatidylinositol (GPI)-anchoring was defective in the pga1(ts) mutants, suggesting that Pga1 is involved in GPI synthesis or its transfer to target proteins. We found GPI18, which was recently reported to encode GPI-mannosyltransferase II (GPI-MT II), as a high-copy suppressor of the temperature sensitivity of pga1(ts). Both Gpi18 and Pga1 were detected in the ER by immunofluorescence, and they were coprecipitated from the Triton X-100-solubilized membrane. The gpi18(ts) and pga1(ts) mutants accumulated the same GPI synthetic intermediate at the restrictive temperature. From these results, we concluded that Pga1 is an additional essential component of the yeast GPI-MT II.