Exploring Endoplasmic Reticulum-Associated Rab GTPases

我们实验室的首要研究项目是表征导致膜接触位点（MCS）形成的机制，两个细胞器之间的接触。我们项目的一个方面是线粒体相关膜（MAM）的表征，这是内质网（ER）和线粒体之间的连接。我们的实验室已经确定了MAM信号机制，MAM靶向基序和决定MAM富集的因素。例如，我们的研究发现小GTPase Rab32是MAM富集和内质网与线粒体之间Ca2+通量的决定因素。Rab32是rabb蛋白的一员，是蛋白质分泌的重要调节因子，其中大约有十几个定位于内质网。在这里，这些Rabs具有多种功能，其中一些具有很好的特征，类似于晚期分泌途径Rabs的“地址标签”功能，而其他Rabs的功能仍然知之甚少。所有这些内质网相关的Rabs的共同之处在于它们在不同程度上通过各种方式决定内质网蛋白在膜结构域的定位。***由于内质网膜结构域的蛋白质组成和内质网膜的定向管化对于内质网与其他细胞器的相互作用至关重要，我们假设内质网相关的Rabs在MCS的形成中起作用。我们的实验室最近对er定位的Rab32亲缘关系的研究结果支持了这一假设。这些蛋白不仅决定内质网蛋白在整个网内的分布，还决定内质网与线粒体的相互作用，并将断裂点定位到线粒体上，调节线粒体动力学。我们将以以下具体目标来检验我们的假设：***1。确定所有人内质网定位Rab蛋白与内质网关联的程度和机制。在内源性和转染Rabs的ER关联量化后，我们将从ER定位和ER排除的Rabs中产生嵌合体，以确定ER定位的机制。* * * 2。内质网相关Rabs在内质网膜接触部位的作用。我们将测试显性活性和显性阴性er定位的Rabs是否可以错定位已知的MCS蛋白。我们还将检测它们影响MCS功能的能力。* * * 3。鉴定er相关Rabs的效应器。我们将使用基于候选的方法来测试er相关的Rabs和已知参与MCS形成的er成型酶之间的相互作用。我们还将通过筛选优先结合gtp结合Rabs的相互作用物来寻找未知的MCS效应物。我们希望这个提案能继续我们在MCS上的研究项目。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。我们还将继续努力，为高素质人才提供一流的培训。**

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). **