Characterization of Retrograde Trafficking at the Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)

内质网-高尔基中间室 (ERGIC) 逆行运输的特征

• 批准号: RGPIN-2018-04833
• 负责人: Ngsee, Johnny
• 金额: $ 2.33万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684201
• 关键词: Characterization; Retrograde; Trafficking; Endoplasmic; Reticulum

Unlike yeast, mammalian endoplasmic reticulum (ER) vesicles do not go directly to the Golgi, but fuse with one another to form the ER-Golgi intermediate compartment (ERGIC). A second trafficking step from the ERGIC is required to reach the Golgi. Likewise, retrograde cargoes from the Golgi transit through the ERGIC en route to the ER. We found that VAPB (VAMP-Associated Protein B) regulate retrograde traffic flow from the ERGIC. Moreover, we found that nuclear envelope (NE) proteins also transit through the ERGIC with VAPB mediating their final delivery to the NE. This discovery provides a unique opportunity to (1) determine the molecular machinery mediating retrograde traffic to the NE, and (2) define the cellular consequences of disrupting retrograde traffic on nucleocytoplasmic transport.****** In Aim 1, we hypothesize that membrane phosphatidylinositol 4-phosphate (PI4P) levels play a permissive role in retrograde ERGIC vesicle formation. PI4P levels determine directional flow of cargoes with high PI4P favoring anterograde traffic to the Golgi and low PI4P driving retrograde flow to the ER. We will use an in vitro reconstituted budding assay to test if changing PI4P levels drives retrograde ERGIC vesicle formation. In our second sub-aim, we will validate that the unconventional SNARE YKT6 mediate fusion of retrograde vesicles with the NE. We will also use the readily detectable NE phenotype we discovered to identify molecules mediating NE fusion. Together, this is a systematic approach to defining the molecular machinery mediating vesicle traffic to the NE.****** Aim 2 examines how disrupting retrograde traffic to the NE affects the permeability of nuclear pores. We found that peripheral nucleoporins are pre-assembled in cytoplasmic membrane vesicles that depend on VAPB for final delivery to the NE. Disrupting this step will alter the composition of nuclear pores and consequently nucleocytoplasmic trafficking of proteins. We will use the import rate of glucocorticoid receptor as an indicator of the permeability status of nuclear pores. GFP-tagged glucocorticoid receptor is rapidly imported into the nucleus upon steroid binding, and the rate of import reflects its passage through the nuclear pores. We will also use TDP-43 and C9orf72 as examples of proteins that depend on nucleocytoplasmic traffic. Both are implicated in premature neuronal loss when disrupted. We expect defective nuclear pore will lead to cytoplasmic relocation of TDP-43 and loss of its nuclear function. For C9orf72, expansion of G4C2 repeats results in translation of dipeptide repeats. We hypothesize that expression of these dipeptide repeats disrupt nucleocytoplasmic traffic by directly plugging the nuclear pores.

与酵母不同，哺乳动物内质网（ER）囊泡不直接进入高尔基体，而是彼此融合形成ER-高尔基体中间室（ERGIC）。需要从ERGIC进行第二步贩运才能到达高尔基体。同样地，逆行的货物从高尔基体经过ERGIC到达ER。我们发现VAP B（VAMP-Associated Protein B）调节ERGIC的逆行交通流。此外，我们发现核膜（NE）蛋白也通过ERGIC转运，VAPB介导它们最终递送到NE。这一发现提供了一个独特的机会，以（1）确定介导逆行运输到NE的分子机制，（2）确定破坏逆行运输对核质运输的细胞后果。在目的1中，我们假设膜磷脂酰肌醇4-磷酸（PI 4P）水平在逆行ERGIC囊泡形成中起着允许的作用。PI 4P水平决定了货物的方向性流动，高PI 4P有利于向高尔基体的顺行运输，低PI 4P驱动向ER的逆行流动。我们将使用体外重构出芽测定来测试改变PI 4P水平是否驱动逆行ERGIC囊泡形成。在我们的第二个子目标中，我们将验证非常规SNARE YKT 6介导逆行囊泡与NE的融合。我们还将使用我们发现的易于检测的NE表型来鉴定介导NE融合的分子。总之，这是一种系统的方法来定义介导囊泡运输到NE的分子机制。目的2研究如何破坏逆行交通NE影响核孔的渗透性。我们发现，外周核孔蛋白预先组装在细胞质膜囊泡，依赖于VAPB的最终交付的NE。破坏这一步骤将改变核孔的组成，从而改变蛋白质的核质运输。我们将用糖皮质激素受体的输入率作为核孔通透性状态的指标。GFP标记的糖皮质激素受体在类固醇结合后迅速输入到细胞核中，并且输入速率反映了其通过核孔的通道。我们还将使用TDP-43和C9 orf 72作为依赖于核质运输的蛋白质的例子。两者都涉及过早的神经元损失时，中断。我们预期缺陷的核孔将导致TDP-43的胞质移位和其核功能的丧失。对于C9 orf 72，G4 C2重复序列的扩增导致二肽重复序列的翻译。我们推测这些二肽重复序列的表达通过直接堵塞核孔来破坏核质运输。