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位点（MCS）形成的机制，即两个细胞器之间的接触。我们的计划的一个方面是表征的内质网（ER）和线粒体之间的连接的线粒体相关膜（MAM）。我们的实验室已经确定了MAM信号传导机制，MAM靶向基序和决定MAM富集的因素。例如，我们的研究已经发现小GTTRab 32作为MAM富集和ER和线粒体之间的Ca 2+通量的决定因素。Rab 32是Rab蛋白的成员，Rab蛋白是蛋白分泌的重要调节因子，其中约有十几个定位于ER。在这里，这些Rabs执行的功能多样性，一些很好地表征和类似的“地址标签”功能的Rabs的后期分泌途径，而其他人执行的功能仍然知之甚少。所有这些ER相关Rab的共同点是，它们在不同程度上通过各种方式决定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膜的定向微管对于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Rabs在MCS的形成中发挥作用。支持这一假设来自我们实验室最近对ER定位Rab 32亲属的发现。这些蛋白质不仅决定了内质网蛋白在整个内质网中的分布，而且决定了内质网与线粒体的相互作用，并将裂隙点定位到线粒体上以调节线粒体动力学。 我们将通过以下具体目标来检验我们的假设： 1.确定所有人类ER定位的Rab蛋白的ER缔合的程度和机制。在定量内源性和转染的Rab的ER-缔合后，我们将从ER-定位和ER-排除的Rab产生嵌合体以确定负责ER定位的机制。 2. ER相关Rabs对ER膜接触位点的作用。我们将测试显性活性和显性阴性ER定位的Rab是否可以错误定位已知的MCS蛋白。我们还将测定它们影响MCS功能的能力。 3.识别ER相关Rab的效应子。我们将使用候选人为基础的方法，将测试ER相关的Rabs和ER成形酶之间的相互作用，已知参与MCS的形成。我们还将通过筛选优先结合GTP结合Rab的相互作用物来寻找未知的MCS效应物。 我们希望这项建议能继续我们对MCS的研究计划。我们将深入了解MCS的功能，并表征一个新的MCS调节蛋白家族。我们也将继续努力，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).