Role of Rab Proteins in Golgi Apparatus Structure and Function

描述（由申请人提供）：高尔基体相关的 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、维持和遗传的关键但仍知之甚少的方面。一般（即“教科书”）范式认为，高尔基体 Rab 蛋白通过招募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颖机制。我们筛选了 Rab33b 和 Rab6（我们的工作涉及高尔基体内部 Rab 级联的两个高尔基体 Rab）与假定的系链蛋白 COG 和 ZW10/RINT-1（逆行高尔基体运输的核心）之间的分子遗传关系。我们发现 Rab6 和 Rab33b 都在系链复合物的上游而不是通过系链复合物起作用。通过电子显微镜断层扫描 (ET)，我们观察到 Rab6 敲低导致高尔基体囊泡出芽/运输明显受到抑制，包被和未包被的出芽轮廓/囊泡沿高尔基池膜的明显积累证明了这一点。这使我们得出结论，Rab6 是有效高尔基体囊泡释放/分裂所必需的。总之，我们的结果表明，Rab6 和 Rab33b 等高尔基体 Rab 在与囊泡分裂相关的高尔基体非常早期的事件中发挥着重要作用，并且它们在这一基本过程中在系链因子招募的上游发挥作用。 因此，根据我们的初步研究，我们认为高尔基体相关 Rab（例如 Rab6 和 Rab33b）的关键且功能上重要的作用是通过上下文敏感的效应器集来调节高尔基体池中不同类别的运输囊泡的出芽/释放。从功能上讲，我们预测每个囊泡类别都支持不同的运输途径，因此在组成上是不同的。在细胞方面，我们假设脑池 Rab 蛋白（例如 Rab6 和可能的 Rab33b）在高尔基体稳态中发挥重要/核心作用。我们的发现支持了这一假设，即与对照细胞相比，Rab6 耗尽的细胞可重复地显示每堆高尔基体池数量显着增加（分别为 4.2 ± 0.32 与 6.8 ± 0.46）。我们认为 Rab 蛋白调节高尔基体驻留蛋白在池之间的分布。我们预测，在 Rab 敲低实验中，单个高尔基体酶的分布将顺式或反式移动。从机制上讲，我们假设不同的效应器，包括已经确定的候选蛋白和其他新蛋白，将调节单个囊泡类别的出芽。我们提出以下三个具体目标来检验这些假设。具体目标 1. 我们将检验以下假设：高尔基体相关 Rab 蛋白（例如 Rab6 和 Rab33b）调节高尔基体池中多种不同类别囊泡的形成和/或释放。具体目标 2. 我们将检验以下假设：高尔基体相关 Rab 调节高尔基体驻留蛋白在池之间的分布。具体目标 3. 我们将测试以下假设：机械上不同的蛋白质组（即效应子）调节单个囊泡类别的出芽/释放。 哺乳动物高尔基体及其调节途径内结构/功能关系的表征对人类健康很重要。我们和其他研究人员发现，此类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部件参与病毒感染和衰老等过程。高尔基体相关 Rab 的调节可能被证明是一种重要的治疗方法。 公共卫生相关性：高尔基体是人体细胞分泌途径中的中心细胞器。如何调节这种细胞器的结构和功能对于细胞的健康乃至人类的健康至关重要。我们发现 Rab 蛋白（Ras 超家族的小 GTP 酶）可能在囊泡形成/释放/运输水平上调节高尔基体组织，这是一个令人惊讶的结果。高尔基体相关 Rab 蛋白功能的缺陷已被证明会影响视力（青光眼）、神经退行性疾病、衣原体感染、病毒进入和衰老。通过高尔基体的逆行运输可用作药物和抗原递送的门户。通过小分子调节 Rab 可能是控制感染和其他疾病问题的一种新方法。了解 Rab 蛋白在人类高尔基体结构和功能关系中的作用确实应该进一步加深我们对人类健康的理解。

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. PUBLIC HEALTH RELEVANCE: The Golgi apparatus is the central organelle within the secretory pathway of human cells. How the structure and function of this organelle is regulated is central to the health of cells and hence to the health humans. We find that Rab proteins, small GTPases of the Ras superfamily, regulate Golgi organization, likely at the level of vesicle formation/release/transport, a surprising outcome. Defects in Golgi associated Rab protein function have been already shown to affect vision (glaucoma), neurodegenerative disease, Chlamydial infection, viral entry and aging. Retrograde trafficking through the Golgi apparatus can be used as a portal for drug and antigen delivery. Rab modulation by small molecules may be a novel approach to controlling infection and other disease problems. Understanding the role of Rab proteins in structure and function relationships within the human Golgi apparatus should indeed further our understanding of human health.