GTP-BINDING PROTEINS AND ENDOCYTOSIS

RAS样GTP结合蛋白组成一个越来越大的家族 相关蛋白，其中许多发挥关键作用，但迄今效果不佳 了解在细胞内膜运输中的作用。在动物细胞中， Rab蛋白家族的成员因其 具有不同细胞器的异质性和特征关联性。 已经在内吞途径上鉴定了几种Rab蛋白， 定位于早期或晚期的内小体。我们最近的结果是 研究表明，早期内小体相关蛋白Rab4控制着 受体循环途径中的重要调控步骤，可能 在受体进入囊泡和小管的过程中 介于早期内体和质膜之间的中间体。既然我们 也表明rab4是p34cdc2磷酸化的底物。 ，它也可能至少部分地对逮捕 有丝分裂细胞的内吞作用。本提案中描述的实验包括 旨在精确地定义Rab4在内胞中的功能 途径，并了解细胞周期依赖的调控机制 膜的流量。首先，我们将通过一个详细的 野生型基因过表达的表型分析 转染细胞中的类型和突变型Rab4。这些实验将 包括完整细胞的分析和无细胞的发展 体外重建Rab4依赖功能的实验。第二，我们 会将Rab4的功能与第二个早期内小体相关的 Rab蛋白Rab5。这项工作将涉及两个Rab的共同表达 蛋白以及对Rab4/rab5嵌合体的分析。第三，到 建立Rab4的作用机制，我们将识别、分离和 鉴定与之相互作用的膜蛋白和胞质蛋白 拉比4。最后，我们将确定磷酸化和 有丝分裂细胞中Rab4与内体的解离是有功能的 与有丝分裂细胞的内吞作用停止有关。使用单元格 用缺乏p34cdc激酶磷酸化的Rab4突变体转染 将分析部位、内吞作用、循环和细胞器组织 使用生化分析和脂质的实时成像相结合- 共聚焦显微镜标记内吞细胞器。这些实验 还将提供有关生物化学的基本信息 有丝分裂细胞中内小体的修饰。

Ras-like GTP binding proteins comprise an increasingly large family of related proteins, many of which play critical but as yet poorly understood roles in intracellular membrane traffic. In animal cells, members of the rab protein family are of particular interest due to their heterogeneity and characteristic associations with distinct organelles. Several rab proteins have been identified on the endocytic pathway, localizing to either early or late endosomes. Our recent results have demonstrated that the early endosome-associated protein rab4 controls an important regulatory step in the pathway of receptor recycling, possibly in the entry of receptors into vesicles and tubules that are intermediates between early endosomes and the plasma membrane. Since we have also shown that rab4 is a substrate for phosphorylation by p34cdc2 kinase, it may also be at least partly responsible for the arrest of endocytosis in mitotic cells. Experiments described in this proposal are designed to precisely define the function of rab4 on the endocytic pathway and to understand the mechanisms of cell cycle-dependent control of membrane traffic. First, we will evaluate rab4 function by a detailed analysis of the phenotypes associated with the overexpression of wild type and mutant rab4 in transfected cells. These experiments will involve the analysis of intact cells and the development of cell-free assays to reconstitute rab4-dependent functions in vitro. Second, we will compare the function of rab4 with a second early endosome-associated rab protein rab5. This work will involve co-expression of both rab proteins as well as an analysis of rab4/rab5 chimera. Third, to establish the mechanism of rab4 action, we will identify, isolate, and characterize both membrane and cytosolic proteins that interact with rab4. Finally, we will determine whether phosphorylation and dissociation of rab4 from endosomes in mitotic cells is functionally related to the arrest of endocytosis in mitotic cells. Using cells transfected with rab4 mutants that lack the p34cdc kinase phosphorylation site, endocytosis, recycling, and organelle organization will be analyzed using a combination of biochemical assays and real-time imaging of lipid- labeled endocytic organelles by confocal microscopy. These experiments will also provide basic information concerning the biochemical modifications of endosomes in mitotic cells.