A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae
A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m205564200
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发表时间：
2002-10-04
影响因子：
4.8
通讯作者：
Umeda, M
中科院分区：
文献类型：
--
作者：
Kato, U;Emoto, K;Umeda, M
Ro 09 -0198（Ro）是一种四环肽抗生素，可特异性结合磷脂酰乙醇胺（PE）并导致细胞溶解。为了研究PE在生物膜中跨双层运动的分子基础，我们分离了一系列对Ro肽高度敏感的芽殖酵母突变体。最敏感的突变体之一，指定为ros 3（RO敏感3），没有表现出显着的变化，在细胞的磷脂成分或在敏感性阿替霉素B，甾醇结合多烯大环内酯类抗生素。这些结果表明ros 3的突变影响质膜上的PE组织，而不是PE合成或膜结构的整体组织。通过功能互补筛选，我们确定了突变体中受影响的基因ROS 3，并且我们表明过敏表型是由ROS 3基因产物Ros 3 p的缺陷表达引起的，Ros 3 p是一种进化上保守的蛋白质，具有两个推定的跨膜结构域。ROS 3基因的破坏导致PE和磷脂酰胆碱的荧光标记类似物的内化显著减少，而荧光标记的磷脂酰丝氨酸和内吞标记物的摄取不受影响。无论是表达水平还是活性的ATP结合盒转运的ros 3Delta细胞不同于野生型细胞，这表明Ros 3 p是不相关的多药耐药活动。结构和亚细胞定位的免疫化学分析表明，Ros 3 p是一种糖基化的膜蛋白，定位于质膜和内质网，并且Ros 3 p的一部分与detergentin可溶性糖脂富集复合物相关。这些结果表明，Ros 3 p是一种膜糖蛋白，在磷脂跨质膜转运中起重要作用。
Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Delta cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergentin-soluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.