Role of Rab Proteins in Golgi Apparatus Structure and Function

Rab 蛋白在高尔基体结构和功能中的作用

• 批准号: 8040313
• 负责人: Brian Storrie
• 金额: $ 26.88万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040313
• 关键词: Address; Affect; Aging; Antigens; Biogenesis; Biological Assay; Cell Fractionation; Cells; Chlamydia Infections; Complex; Defect; Disease; Electron Microscope; Electron Microscopy; Enzymes; Event; Glaucoma; Golgi Apparatus; Health; Homeostasis; Human; In Situ; Individual; Infection Control; Lead; Maintenance; Mediating; Membrane; Membrane Fusion; Molecular Genetics; Monomeric GTP-Binding Proteins; Neurodegenerative Disorders; Organelles; Outcome; Pathway interactions; Pharmaceutical Preparations; Phenotype; Play; Process; Protein Isoforms; Proteins; RNA Interference; Reagent; Regulatory Pathway; Relative (related person); Research Personnel; Role; Screening procedure; Set protein; Small Interfering RNA; Structure; Structure-Activity Relationship; Sum; Testing; Textbooks; Therapeutic; Transport Vesicles; Vesicle; Viral; Virus Diseases; Vision; Work; base; cell killing; cell type; human disease; in vitro Assay; novel; novel strategies; protein function; research study; small molecule; tomography; trafficking; ward

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.

描述（由申请人提供）：高尔基体相关Rab蛋白- Ras超家族的小gtpase -通过影响囊泡运输调节高尔基体生物发生，维持和遗传的关键但鲜为人知的方面。一般的（即“教科书”）范式认为，高尔基氏Rab蛋白通过募集和激活介导膜融合初始步骤的系缚因子来调节囊泡靶向。我们的初步研究支持一种不同的、新颖的机制。我们筛选了Rab33b和Rab6之间的分子遗传关系（Rab33b和Rab6是我们的研究表明在高尔基拉布级联中的两种高尔基拉布），以及推测的系链蛋白COG和ZW10/ rrt -1，它们是逆行高尔基转运的核心。我们发现Rab6和Rab33b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Rab6的下调导致高尔基体囊泡出芽/运输的明显抑制，这可以通过高尔基池膜上涂层和未涂层出芽轮廓/囊泡的明显积累来证明。这使我们得出结论，Rab6是有效的高尔基囊泡释放/分裂所必需的。总之，我们的研究结果表明，Rab6和Rab33b等高尔基Rabs在与囊泡分裂相关的高尔基体早期事件中起着重要作用，并且在这一基本过程中，它们在系绳因子募集的上游起作用。因此，基于我们的初步研究，我们认为高尔基相关Rabs（如Rab6和Rab33b）的一个关键和功能上的重要作用是通过上下文敏感效应集调节高尔基池中不同类型运输囊泡的出芽/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。在细胞上，我们假设池中Rab6和Rab33b等rabb蛋白在高尔基体稳态中发挥重要/核心作用。我们的研究结果支持了这一假设，与对照细胞相比，Rab6缺失的细胞可重复地显示每堆叠高尔基池的数量显著增加（分别为4.2 + 0.32和6.8 + 0.46）。我们认为Rab蛋白调节了高尔基驻留蛋白在池池之间的分布。我们预测，在Rab敲低实验中，单个高尔基酶的分布将顺式或反式转移。在机制上，我们假设不同的效应物，包括已经确定的候选蛋白质和其他新的，将调节单个囊泡类的萌芽。我们提出以下三个具体目标来检验这些假设。具体目标我们将验证高尔基相关的Rab6和Rab33b蛋白调节高尔基池中多种不同类型囊泡的形成和/或释放的假设。具体目标2。我们将检验高尔基相关Rabs调节池之间高尔基驻留蛋白分布的假设。具体目标3。我们将测试这一假设，即机制上不同的蛋白质组，即效应物，调节单个囊泡类的出芽/释放。表征哺乳动物高尔基体及其调控途径内的结构/功能关系对人类健康至关重要。我们和其他研究人员发现，这些途径是进入细胞的有用门户，用于传递细胞杀伤试剂和抗原。这些途径中的缺陷可导致人类疾病。此外，这些途径中的重要机械成分还参与病毒感染和衰老等过程。调节高尔基相关Rabs可能是一种重要的治疗方法。