Characterization of mammalian COG complex-interacting Golgi trafficking machinery

项目摘要/摘要 高尔基体内的囊泡转运是研究靶向特异性的有用系统。 细胞内运输小泡。哺乳动物高尔基体由四到八个池子组成，每个池子 含有一套独特的蛋白质和脂肪修饰酶。通过高尔基山脉的大部分运输发生在 通过脑池成熟，这表明分泌蛋白总是留在脑池里，而 驻留的酶通过逆行小泡循环。这种排序模型意味着几种不同类型的 逆行小泡(具有不同的酶含量)产生高尔基体不同的酶分布。 高尔基体逆行小泡系留的中心协调者是进化上保守的 寡聚高尔基体(COG)复合体。COG复合体由八种基因产物组成，每一种都是至关重要的 为高尔基人的活动。COG复合体在高尔基体糖基化小泡连接中的作用 驻留在顺式、中间和反式隔室的酶。因此，7个COG亚基的缺陷 在先天性II型糖基化障碍患者中被发现。COG复合体是一种 广泛的基本过程，包括蛋白质和脂肪糖基化、分选和逆行囊泡 但COG的确切作用机制仍是个谜。HEK293T和HeLa的详细分析 缺失COG亚基的敲除(KO)细胞系表明，每个亚基都是不可或缺的 整个COG综合体的稳定性和功能。此外，一个完整的COG综合体对于 高尔基酶的一个子集的稳定性，但对另一个子集不是必需的，表明两者都存在 COG依赖和COG非依赖的高尔基体循环途径。我们认为，COG情结 通过与特定陷阱的多管齐下的相互作用，协调高尔基酶的子集的局部循环， RABS和盘绕线圈系绳系数。COG故障导致COG迅速损失和降解。 依赖小泡，触发选择性代偿机制的上调(胆固醇的调节 生物合成、高尔基体核周重新定位和内吞途径的修饰)，这些都是必不可少的 用于COG缺陷的人类细胞的生存。为了验证这一假设，我们首先将利用经过基因编辑的细胞系和 结合生化和显微镜工具来研究COG复合体的分子细节- 依赖酶循环途径和COG复合体非依赖途径(目标1)。接下来，我们将 体外重建COG复合体的囊泡拴系活性(目标2)。最后，我们将研究新的压力和 允许COG剥夺的人类细胞存活的代偿机制(目标3)。 几个独立的高尔基再循环机制的存在可能对弹性很重要 真核生物的分泌和内吞途径。理解COG复合体是如何在空间上和 在时间上控制选定运输囊泡的精确拴系对于我们理解 人类细胞的膜转运和蛋白质糖基化。

PROJECT SUMMARY/ABSTRACT Vesicle transport within the Golgi apparatus is a useful system for studying the targeting specificity of intracellular transport vesicles. The mammalian Golgi consists of four to eight cisternae, each of these containing a unique set of protein- and lipid-modifying enzymes. Most of the transport through the Golgi occurs by cisternal maturation, which suggests that secretory proteins always remain within the cisternae, while resident enzymes are recycled via retrograde vesicles. This sorting model implies that several distinct types of retrograde vesicles (with differing enzyme content) generate the Golgi’s differential enzyme distribution. The central coordinator for retrograde vesicle tethering at the Golgi is the evolutionarily conserved oligomeric Golgi (COG) complex. The COG complex consists of eight gene products, each of which is critical for the Golgi functions. The COG complex functions in the tethering of vesicles that recycle Golgi glycosylation enzymes residing in cis, medial, and trans compartments. Consequently, defects in seven COG subunits have been identified in patients with congenital disorders of glycosylation type II. The COG complex is required for a broad range of essential processes, including protein and lipid glycosylation, sorting and retrograde vesicular trafficking, but the exact mechanism of COG function is an enigma. Detailed analyses of HEK293T and HeLa knock-out (KO) cell lines depleted of individual COG subunits have revealed that each subunit is indispensable to the stability and function of the entire COG complex. Further, a complete COG complex is essential for the stability of a subset of Golgi enzymes, but nonessential for another subset, indicating the existence of both COG-dependent and COG-independent Golgi recycling pathways. We propose that the COG complex orchestrates local recycling of a subset of Golgi enzymes via multipronged interaction with specific SNAREs, Rabs, and coiled-coil tethering factors. COG malfunction results in a rapid loss and degradation of COG- dependent vesicles, triggering upregulation of selective compensatory mechanisms (modulation of cholesterol biosynthesis, Golgi perinuclear repositioning, and modification of the endocytic pathway) which are essential for survival of COG-deficient human cells. To test this hypothesis, first we will utilize gene-edited cell lines and a combination of biochemical and microscopy tools to investigate the molecular details of the COG complex– dependent enzyme-recycling pathway and the COG complex–independent pathway (Aim 1). Next, we will reconstitute COG complex vesicle-tethering activity in vitro (Aim 2). Finally, we will investigate novel stress and compensatory mechanisms that allow for the survival of COG-deprived human cells (Aim 3). The existence of several independent Golgi recycling mechanisms is likely to be important for the elasticity of eukaryotic secretory and endocytic pathways. Understanding how the COG complex spatially and temporally controls the precise tethering of selected transport vesicles is critical to our understanding of membrane trafficking and protein glycosylation in human cells.