Protein-protein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis
Protein-protein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis
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DOI：
10.1073/pnas.112202799
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发表时间：
2002-07-23
影响因子：
11.1
通讯作者：
Bard, M
中科院分区：
文献类型：
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作者：
Mo, C;Valachovic, M;Bard, M
一项酿酒酵母微阵列表达研究表明，ORF YER044C（现命名为ERG28）与麦角甾醇生物合成密切相关。ERG28基因的破坏导致与erg26和erg27缺失菌株相似的甾醇中间体的缓慢生长和积累，这表明Erg28p可能与Erg26p和/或Erg27p相互作用。本研究通过在ERG26和ERG27基因上添加人血凝素蛋白（HA）表位标签的肽段，在ERG28基因上添加Myc标签，检测Erg28p和Erg26p/Erg27p之间的相互作用。差速离心显示Erg26p、Erg27p和Erg28p均为膜相关蛋白。绿色荧光蛋白融合蛋白定位研究表明，Erg26p、Erg27p和Erg28p均位于内质网。用兔抗Erg25p进行的溶解膜蛋白共免疫沉淀研究表明，Erg25p可与Erg27p和Erg28p共免疫沉淀。Erg28p也被证明与Erg27p相互共免疫沉淀。然而，没有观察到与Erg26p的共免疫沉淀，很可能是因为该蛋白的溶解性差。蔗糖梯度超离心研究表明，Erg25p/Erg26p/Erg27p/Erg28p与其他甾醇生物合成蛋白可能形成66 - 200 kDa的复合物。使用以Erg27p- ha和Erg26p- ha为靶蛋白的抗ha柱，鉴定出含有Erg25p/Erg26p/Erg27p/Erg28p的复合物。因此，我们认为Erg28p作为跨膜支架连接Erg27p和其他CA去甲基化蛋白（Erg25p, Erg26p），在内质网形成去甲基化复合物。
A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis. Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and/or Erg27p. In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p/Erg27p. Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins. Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum. Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p. Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p. However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein. Sucrose gradient ultracentrifugation studies suggested that Erg25p/Erg26p/Erg27p/Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa. Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p/Erg26p/Erg27p/Erg28p was identified. Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other CA demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.