Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae
Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae
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DOI：
10.1091/mbc.e02-09-0609
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发表时间：
2003-03-01
影响因子：
3.3
通讯作者：
Powers, T
中科院分区：
文献类型：
--
作者：
Wedaman, KP;Reinke, A;Powers, T
Tor1p和Tor2p激酶是免疫抑制抗生素雷帕霉素的靶标，是一个高度保守的信号网络的组成部分，该网络将营养可用性和细胞生长结合在一起。为了深入了解tor依赖信号的分子基础，我们使用细胞分离和免疫亲和层析来检测Tor2p的物理环境。我们发现大部分Tor2p与至少四种其他蛋白（Avo1p-Avo3p和Lst8p）一起与膜结合的隔室结合。利用免疫金电镜，我们观察到Tor2p和Tor1p以点状簇的形式定位于靠近质膜和细胞内部的区域，通常与特征性的膜迹有关。细胞分离、共免疫沉淀和免疫金电镜实验证实，Lst8在这些膜位点与Tor2p和Tor1p结合。相反，我们发现哺乳动物Tor调控蛋白Raptor的酵母同源物Kog1优先与Tor1p相互作用。这些发现为Tor信号复合物的存在提供了证据，这些复合物包含不同的以及重叠的成分。这些复合物共定位于膜结合的隔室，这表明膜介导的信号传导与Tor活性之间存在密切关系。
Tor1p and Tor2p kinases, targets of the immune-suppressive antibiotic rapamycin, are components of a highly conserved signaling network that couples nutrient availability and cell growth, To gain insight into the molecular basis underlying Tor-dependent signaling, we used cell fractionation and immunoaffinity chromatography to examine the physical environment of Tor2p. We found that the majority of Tor2p associates with a membrane-bound compartment along with at least four other proteins, Avo1p-Avo3p and Lst8p. Using immunogold electron microscopy, we observed that Tor2p, as well as Tor1p, localizes in punctate clusters to regions adjacent to the plasma membrane and within the cell interior, often in association with characteristic membranous tracks. Cell fractionation, coimmunoprecipitation, and immunogold electron microscopy experiments confirmed that Lst8 associates with both Tor2p as well as Tor1p at these membranous sites. In contrast, we find that Kog1, the yeast homologue of the mammalian Tor regulatory protein Raptor, interacts preferentially with Tor1p. These findings provide evidence for the existence of Tor signaling complexes that contain distinct as well as overlapping components. That these complexes colocalize to a membrane-bound compartment suggests an intimate relationship between membrane-mediated signaling and Tor activity.