Role of Rab Proteins in Golgi Apparatus Structure and Function

描述（由申请人提供）：高尔基体相关Rab蛋白-Ras超家族的小GTP酶-通过影响囊泡运输来调节高尔基体生物发生、维持和遗传的关键方面，但知之甚少。一般（即，“教科书”）范式认为，高尔基体Rab蛋白通过募集和激活介导膜融合初始步骤的束缚因子来调节囊泡靶向。我们的初步研究支持一种不同的新机制。我们筛选了Rab 33 b和Rab 6之间的分子遗传关系-我们的工作涉及高尔基体内Rab级联反应的两个高尔基体Rab-以及推定的系链蛋白COG和ZW 10/RINT-1，对高尔基体逆行运输至关重要。我们发现Rab 6和Rab 33 b都作用于系链复合物的上游，而不是通过系链复合物。通过电子显微镜断层扫描（ET），我们观察到Rab 6敲低导致高尔基体囊泡出芽/运输的明显抑制，如由涂覆和未涂覆的出芽轮廓/囊泡沿着高尔基体池膜的显著积累所证明的。这使我们得出结论，Rab 6是有效的高尔基体囊泡释放/切断所必需的。总之，我们的研究结果表明，高尔基Rabs，如Rab 6和Rab 33 b服务的重要功能，在非常早期的事件在高尔基体相关的囊泡断裂，他们的上游的系链因子招聘在这个基本的过程。 基于我们的初步研究，我们认为，高尔基体相关的Rabs（如Rab 6和Rab 33 b）的一个关键的和功能上重要的作用是调节，通过上下文敏感的效应集，从高尔基体池的运输囊泡的不同类别的出芽/释放。在功能上，我们预测每个囊泡类支持不同的运输途径，因此在组成上是不同的。细胞，我们假设，脑池Rab蛋白，如Rab 6和可能Rab 33 b发挥重要/核心作用，高尔基体内平衡。这一假设得到了我们的发现的支持，与对照细胞相比，Rab 6耗尽的细胞可重复地显示每个堆叠的高尔基池数量显著增加（分别为4.2 ± 0.32对6.8 ± 0.46）。我们建议，Rab蛋白调节高尔基常驻蛋白的分布之间的脑池。我们预测，在Rab敲除实验中，单个高尔基体酶的分布将被顺式或反式移动。从机制上讲，我们假设不同的效应物，包括已经确定的候选蛋白和其他新的，将调节个别囊泡类的萌芽。我们提出以下三个具体目标来检验这些假设。具体目标1。我们将测试的假设，高尔基体相关的Rab蛋白，如Rab 6和Rab 33 b调节的形成和/或释放的多个，不同类别的囊泡从高尔基体池。具体目标2。我们将测试的假设，高尔基体相关的Rabs调节高尔基体常驻蛋白质之间的分布池。具体目标3。我们将测试的假设，机械不同的蛋白质集，即，效应子，调节单个囊泡类的出芽/释放。 哺乳动物高尔基体及其调控通路的结构/功能关系的表征对人类健康是重要的。我们和其他研究人员已经发现，这些途径是进入细胞的有用门户，用于递送细胞杀伤试剂和抗原。这些途径的缺陷可能导致人类疾病。此外，这些途径中的重要机械组件参与病毒感染和衰老等过程。调节高尔基体相关的Rabs可能被证明是一种重要的治疗方法。

DESCRIPTION (provided by applicant): Golgi associated Rab proteins - small GTPases of the Ras superfamily -- regulate critical, yet poorly understood, aspects of Golgi biogenesis, maintenance, and inheritance through affecting vesicle trafficking. The general (i.e., "textbook") paradigm holds that Golgi Rab proteins regulate vesicle targeting via the recruitment and activation of tethering factors that mediate initial steps of membrane fusion. Our Preliminary Studies support a different and novel mechanism. We screened for molecular genetic relationship(s) between Rab33b and Rab6 - two Golgi Rabs that our works implicates in an intra-Golgi Rab cascade -- and the putative tether proteins, COG and ZW10/RINT-1, central to retrograde Golgi trafficking. We found that both Rab6 and Rab33b acted upstream of, not through, the tether complexes. By electron microscope tomography (ET), we observed that Rab6 knockdown results in an apparent inhibition of Golgi vesicle budding/transport as evidenced by the pronounced accumulation of both coated and uncoated budding profiles/vesicles along Golgi cisternal membranes. This has led us to conclude that Rab6 is required for efficient Golgi vesicle release/scission. In sum, our results suggest that Golgi Rabs such as Rab6 and Rab33b serve important functions in very early events at the Golgi related to vesicle scission, and that they act upstream of tether factor recruitment in this fundamental process. Based on our Preliminary Studies, we thus contend that a critical and functionally important role of Golgi-associated Rabs (such as Rab6 and Rab33b) is to regulate, through context sensitive effector sets, the budding/release of distinct classes of transport vesicles from Golgi cisternae. Functionally, we predict that each vesicle class supports a distinct trafficking pathway(s) and as such are compositionally distinct. Cellularly, we hypothesize that cisternal Rab proteins such as Rab6 and likely Rab33b play an important/central role in Golgi homeostasis. This hypothesis is supported by our finding that compared to control cells, Rab6 depleted cells reproducibly reveal a significant increase in the number of Golgi cisternae per stack (4.2 + 0.32 versus 6.8 + 0.46, respectively). We propose that Rab proteins regulate the distribution of Golgi resident proteins between cisternae. We predict that in Rab knockdown experiments that the distribution of individual Golgi enzymes will be shifted cis- or trans-ward. Mechanistically, we hypothesize that distinct effectors, including already identified candidate protein and others novel, will modulate the budding of individual vesicle classes. We propose the following three Specific Aims to test these hypotheses. Specific Aim 1. We will test the hypothesis that Golgi associated Rab proteins such as Rab6 and Rab33b regulate the formation and/or release of multiple, distinct classes of vesicles from Golgi cisternae. Specific Aim 2. We will test the hypothesis that Golgi-associated Rabs regulate the distribution of Golgi resident proteins between cisternae. Specific Aim 3. We will test the hypothesis that mechanistically distinct protein sets, i.e., effectors, modulate the budding/release of individual vesicle classes. Characterization of structural/functional relationships within the mammalian Golgi apparatus and its regulatory pathways is important to human health. We and other investigators have found that such pathways are useful portals into the cell for the delivery of cell-killing reagents and antigens. Defects in these pathways can lead to human disease. Moreover, important machinery components in these pathways are involved in such processes as virus infection and aging. Modulation of Golgi associated Rabs may prove to be an important therapeutic approach.