Coordination of membrane traffic through rab GEF and GAP cascades

通过 rab GEF 和 GAP 级联协调膜运输

基本信息

批准号：
8508270
负责人：
PETER Jay NOVICK
金额：
$ 33.3万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8508270
关键词：
Address Affect Alleles Antigen Presentation Binding Biological Assay Bypass Candidate Disease Gene Cell Polarity Cell Surface Receptors Cell membrane Cell physiology Complex Diabetes Mellitus Epithelial Cells Feedback Flow-It Fluorescence GTP Binding GTPase-Activating Proteins Genetic screening method Glucose Transporter Golgi Apparatus Guanine Nucleotide Dissociation Inhibitors Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hydrolysis Insulin Kinetics Lead Length Link Maintenance Malignant Neoplasms Mammalian Cell Mediating Membrane Membrane Protein Traffic Modification Molecular Motor Mutation Nerve Degeneration Organelles Pathway interactions Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Preparation Protein Export Pathway Proteins Recombinant Proteins Recruitment Activity Regulation Role SNAP receptor Secretory Vesicles Series Site Sorting - Cell Movement Staging Surface TRAPP transport protein particle Testing Vesicle Work Yeasts human disease member mutant neurotransmission novel phosphatidylinositol 4-phosphate programs rab GTP-Binding Proteins response

项目摘要

DESCRIPTION (provided by applicant): Rabs represent the largest branch of the Ras GTPase superfamily, with ten members in yeast and more than 60 in mammalian cells. They serve as master regulators of membrane traffic, each typically controlling several different aspects of a specific stage of membrane traffic by recruiting diverse effectors proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs, in turn, are regulated by specific guanine nucleotide exchange proteins (GEFs) that catalyze the displacement of GDP and binding of GTP and GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. Recent work from our lab has demonstrated that different Rabs are networked to one another through their regulators. Specifically we have shown that the Rab, Ypt32, in its GTP-bound form recruits Sec2, the GEF that activates the downstream Rab, Sec4, as well as Gyp1, the GAP that inactivates the upstream Rab, Ypt1. The net effect is a programmed series of Rab conversions that lead to changes in the functional identity of the membrane as it flows along the exocytic pathway. We have also shown that the Golgi pool of phosphatidylinositol 4-phosphate (PI4P) works in concert with Ypt32 to recruit Sec2 and to control a regulatory switch in Sec2 function. We propose five specific aims to address the molecular mechanisms by which Rabs are networked to one another and by which the distribution of PI4P is spatially defined. 1. We will explore the role of phosphorylation in the regulation of Sec2 function. Phosphomimetic and non- phosphorylatable alleles will be tested for their interactions with Sec4, Ypt32, Sec15 and PI4P. 2. The Sec4 GEF Sec2 binds to the Sec4 effectors Sec15 leading to a positive feedback loop. To test the role of this mechanism in membrane traffic we will generate Sec2 alleles specifically defective in Sec15 binding. 3. We have generated an allele of Ypt1 that can be activated by the Sec4 GEF, Sec2. We will determine if this mutation redirects the membrane association of Ypt1 and the effects of this "short circuit" on membrane traffic. 4. We have evidence for a second example of a Rab-GAP cascade and will screen all Rab GAPs against representative Rabs to identify more. We will test the effects of GAP disruption on overlap of Rab domains. 5. PI4P is normally restricted to the Golgi and is not enriched on Golgi-derived secretory vesicles. We will determine the mechanism by which PI4P is limited to the Golgi.

描述（由申请人提供）：RABS代表RAS GTPase超家族的最大分支，在酵母中有十名成员，哺乳动物细胞中有60多个成员。它们是膜流量的主要调节剂，每个调节剂通常通过募集多种效应子蛋白（例如细胞骨架电动机，囊泡绑扎蛋白和SNARE COMPLECS组装的调节剂）来控制膜流量的特定阶段的几个不同方面。反过来，RABS受特定的鸟嘌呤核苷酸交换蛋白（GEF）的调节，该蛋白（GEF）催化GDP的位移以及GTP和GTPase激活蛋白（GAPS）的结合，刺激GTP水解的内在速率缓慢。我们实验室的最新工作表明，不同的RABS通过其监管机构彼此联网。具体而言，我们已经证明了RAB YPT32以GTP结合形式募集SEC2，即激活下游RAB，SEC4以及GYP1的GEF，这是使上游RAB，YPT1失活的差距。净效应是一系列编程的RAB转换系列，在膜沿外环途径流动时会导致膜的功能身份变化。我们还表明，与YPT32一起使用的磷脂酰肌醇4-磷酸（PI4P）的高尔基池可募集SEC2并控制SEC2功能中的调节开关。我们提出了五个特定的目的，以解决Rabs彼此联网的分子机制，并通过空间定义PI4P的分布。 1。我们将探讨磷酸化在SEC2功能调节中的作用。磷酸化和非磷酸化等位基因与SEC4，YPT32，SEC15和PI4P的相互作用将测试。 2。SEC4 GEF SEC2与SEC4效应子SEC15结合，导致正反馈循环。为了测试该机制在膜流量中的作用，我们将生成在SEC15结合中特别有缺陷的SEC2等位基因。 3。我们生成了YPT1的等位基因，该等位基因可以被SEC4 GEF Sec2激活。我们将确定该突变是否将YPT1的膜关联重定向以及该“短路”对膜流量的影响。 4。我们有证据表明RAB-GAP级联反应的第二个例子，并将筛选所有针对代表性RAB的RAB差距以识别更多。我们将测试间隙破坏对RAB域重叠的影响。 5。PI4P通常仅限于高尔基体，并且不富含高尔基衍生的分泌囊泡。我们将确定PI4P仅限于高尔基体的机制。