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: 8537212
• 负责人: Brian Storrie
• 金额: $ 31.52万
• 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/8537212
• 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; 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; screening; 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.

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、维持和遗传的关键但知之甚少的方面。一般的(即“教科书”)范式认为，高尔基体Rab蛋白通过重新招募和激活介导膜融合初始步骤的拴系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(S)--我们的工作涉及高尔基体内的两个高尔基体兔--以及可能的系链蛋白COG和ZW10/Rint-1，它们是逆行高尔基体运输的中心。我们发现Rab6和Rab33b都作用于系绳复合体的上游，而不是通过。通过电子显微镜断层扫描(ET)，我们观察到Rab6基因敲除导致高尔基体小泡的萌发/运输受到明显的抑制，这从高尔基体池膜上有明显的有涂层和无涂层的萌发轮廓/囊泡的堆积得到了证明。这使得我们得出结论，Rab6是有效释放/断裂高尔基体小泡所必需的。综上所述，我们的结果表明，Golgi Rabb，如Rab6和Rab33b，在高尔基体与囊泡断裂相关的早期事件中发挥着重要作用，并且它们在这一基本过程中发挥着系绳因子招募的上游作用。因此，基于我们的初步研究，我们认为高尔基体相关的RABS(如Rab6和Rab33b)的一个关键和重要的功能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小泡的萌发/释放。在功能上，我们预测每一类囊泡支持一种不同的运输途径(S)，因此在成分上是不同的。在细胞上，我们假设脑池中的Rab蛋白，如Rab6和可能的Rab33b在高尔基体内稳态中起着重要/中心的作用。这一假说得到了我们的发现的支持，即与对照细胞相比，Rab6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)。我们认为Rab蛋白调节高尔基体驻留蛋白在脑池之间的分布。我们预测，在RAB基因敲除实验中，单个高尔基体酶的分布将向顺式或反式转移。在机制上，我们假设不同的效应器，包括已经识别的候选蛋白质和其他新的，将调节单个囊泡类别的萌发。我们提出了以下三个具体目标来检验这些假说。具体目标1.我们将检验高尔基体相关Rab蛋白(如Rab6和Rab33b)调节高尔基体池中多种不同类别囊泡的形成和/或释放的假设。具体目的2.我们将检验高尔基体相关RABS调节高尔基体驻留蛋白在脑池之间分布的假设。具体目标3.我们将检验这一假设，即机械上不同的蛋白质集，即效应器，调节单个囊泡类别的萌发/释放。哺乳动物高尔基体及其调控途径中的结构/功能关系的表征对人类健康非常重要。我们和其他研究人员发现，这种途径是进入细胞的有用门户，用于运送细胞杀伤剂和抗原。这些通路的缺陷可能会导致人类疾病。此外，这些途径中的重要机械部件参与了病毒感染和衰老等过程。高尔基体相关RABS的调节可能被证明是一种重要的治疗方法。