Characterization of mammalian COG complex-interacting Golgi trafficking machinery

项目概要/摘要 高尔基体内的囊泡转运是用于研究靶向特异性的有用系统， 胞内转运囊泡。哺乳动物的高尔基体由四到八个池组成，每个池 含有一组独特的蛋白质和脂质修饰酶。大部分通过高尔基体的运输发生在 通过脑池成熟，这表明分泌蛋白总是留在脑池内，而 常驻酶通过逆行囊泡再循环。这种排序模型意味着几种不同类型的 逆行囊泡（具有不同的酶含量）产生高尔基体的差异酶分布。 在高尔基体逆行囊泡束缚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，每一种都是关键的 高尔基体的功能。COG复合物在回收高尔基体糖基化的囊泡的束缚中起作用 存在于顺式、中间和反式隔室中的酶。因此，七个COG子单位的缺陷 在患有先天性糖基化II型疾病的患者中发现。需要COG综合体， 广泛的基本过程，包括蛋白质和脂质糖基化，分选和逆行囊泡 贩运，但COG功能的确切机制是一个谜。HEK 293 T和HeLa的详细分析 耗尽单个COG亚基的敲除（KO）细胞系已经揭示每个亚基是不可缺少的 整个COG综合体的稳定性和功能。此外，一个完整的COG复杂的是必不可少的 高尔基体酶的一个子集的稳定性，但对另一个子集不重要，表明两者的存在 依赖于COG和不依赖于COG的高尔基体再循环途径。我们建议COG综合体 通过与特定SNARE的多管齐下的相互作用协调高尔基体酶亚群的局部再循环， Rabs和螺旋线圈拴系因子。COG故障导致COG快速损失和降解- 依赖性囊泡，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，以及内吞途径的修饰），这些都是必不可少的 对于缺乏COG的人类细胞的存活。为了验证这一假设，首先我们将利用基因编辑的细胞系， 生物化学和显微镜工具的组合，以研究COG复合物的分子细节- 依赖性酶循环途径和COG复合物非依赖性途径（Aim 1）。接下来我们就 体外重组COG复合物的囊泡束缚活性（目的2）。最后，我们将研究新的压力， 补偿机制，允许COG-剥夺的人细胞的生存（目的3）。 几个独立的高尔基体回收机制的存在可能是重要的弹性 真核分泌和内吞途径。了解COG如何在空间上和 在时间上控制所选运输囊泡的精确束缚对于我们理解 人细胞中的膜运输和蛋白质糖基化。

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.