Exploring Endoplasmic Reticulum-Associated Rab GTPases

我们实验室的总体研究计划是表征导致膜接触部位(MCS)形成的机制，膜接触部位是两个细胞器之间的接触。我们项目的一个方面是对线粒体相关膜(MAM)的表征，MAM是内质网(ER)和线粒体之间的连接。我们的实验室已经确定了MAM信号转导机制、MAM靶向基序以及决定MAM浓缩的因素。例如，我们的研究发现，小GTP酶Rab32是MAM丰富和内质网和线粒体之间钙离子流量的决定因素。Rab32是Rab蛋白的一员，Rab蛋白是蛋白质分泌的重要调节因子，其中约有12个定位于内质网。在这里，这些RAB执行不同的功能，其中一些很好地描述并类似于晚期分泌途径中RAB的“地址标签”功能，而另一些执行的功能仍然鲜为人知。所有这些与ER相关的RAB的共同之处在于，它们在不同的程度上通过不同的手段确定ER蛋白在膜结构域的定位。 由于ER膜结构域的蛋白质组成和ER膜的定向管状连接对于ER与其他细胞器的相互作用至关重要，因此我们假设ER相关的RAB在MCS的形成中发挥了作用。这一假说的支持来自我们实验室最近在ER定位的Rab32亲属上产生的发现。这些蛋白质不仅决定了ER蛋白在整个网状结构中的分布，还决定了ER与线粒体的相互作用，并将裂点定位在线粒体上，从而调节线粒体的动力学。 我们将通过以下具体目标来验证我们的假设： 1.确定所有人ER定位的Rab蛋白的ER结合程度和机制。在量化内源性和转基因RAB的ER相关性后，我们将从ER定位的RAB和ER排除的RAB中产生嵌合体，以确定负责ER定位的机制。 2.ER相关RABS在ER膜接触位点中的作用我们将测试显性活性和显性负性ER定位的RABS是否可以错误定位已知的MCS蛋白。我们还将分析它们影响MCS功能的能力。 3.确定ER相关RAB的效应器。我们将使用基于候选人的方法，测试ER相关的RABS和已知参与MCS形成的ER成形酶之间的相互作用。我们还将通过筛选优先结合GTP结合的RAB的交互作用来寻找未知的MCS效应器。 我们希望这项提案将继续我们对MCS的研究计划。我们将深入了解MCS的功能，并描述一个新的MCS调节蛋白家族。我们还将继续努力为高素质人才(HQP)提供一流的培训。

The overarching research program of our laboratory is the characterization of mechanisms that lead to the formation of membrane contact sites (MCS), contacts between two organelles. One aspect of our program is the characterization of the mitochondria-associated membrane (MAM), a connection between the endoplasmic reticulum (ER) and mitochondria. Our lab has identified MAM signaling mechanisms, MAM targeting motifs and factors that determine MAM enrichment. For instance, our research has discovered the small GTPase Rab32 as a determinant of MAM enrichment and of the flux of Ca2+ between the ER and mitochondria. Rab32 is a member of the Rab proteins, important regulatory factors for protein secretion, of which about a dozen localize to the ER. Here, these Rabs perform a diversity of functions, some well characterized and resembling the “address tag” function of Rabs of the late secretory pathway, whereas others perform functions that remain poorly understood. What all these ER-associated Rabs have in common is that they determine to varying extents and by various means the localization of ER proteins to membrane domains. Since the protein composition of ER membrane domains and directed tubulation of ER membranes are critical for the interaction of the ER with other organelles, we hypothesize that ER-associated Rabs play a role for the formation of MCS. Support for this hypothesis comes from recent findings generated in our lab on ER-localized Rab32 relatives. These proteins not only determine the distribution of ER proteins within the whole reticulum, but also determine ER-mitochondria interaction and localize fissure points onto mitochondria to regulate mitochondrial dynamics. We will test our hypothesis with the following specific aims: 1. Determine the extent and mechanism of ER association for all human ER-localized Rab proteins. Upon quantification of ER-association for endogenous and transfected Rabs, we will generate chimera from ER-localized and ER-excluded Rabs to determine mechanisms responsible for ER localization. 2. Role of ER-associated Rabs for ER membrane contact sites. We will test whether dominant-active and dominant-negative ER-localized Rabs can mislocalize known MCS proteins. We will also assay for their ability to influence MCS function. 3. Identify effectors for ER-associated Rabs. We will use candidate-based approaches that will test for interaction between ER-associated Rabs and ER-shaping enzymes with known involvement in MCS formation. We will also search for unknown MCS effectors by screening for interactors that preferentially bind GTP-bound Rabs. We expect this proposal to continue our research program on MCS. We will provide insight into the functioning of MCS and characterize a novel family of MCS regulatory proteins. We will also continue our efforts to provide first class training for high quality personnel (HQP).