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

与酵母不同，哺乳动物内质网(ER)小泡不直接到达高尔基体，而是相互融合形成ER-高尔基体中间隔室(ERGIC)。从ERGIC开始的第二个贩运步骤需要到达高尔基。同样，来自高尔基山脉的逆行货物在前往急诊室的途中通过ERGIC过境。我们发现VAPB(VAMP相关蛋白B)调节ERGIC的逆行交通流。此外，我们还发现核膜(NE)蛋白也通过ERGIC传递，VAPB介导它们最终传递到NE。这一发现提供了一个独特的机会来(1)确定介导逆行交通到NE的分子机制，以及(2)确定中断逆行交通对核质运输的细胞后果。*在目标1中，我们假设膜磷脂酰肌醇4-磷酸(PI4P)水平在逆行ERGIC囊泡形成中起允许作用。PI4P水平决定了货物的定向流动，高PI4P有利于顺行交通到高尔基山脉，低PI4P推动逆流到ER。我们将使用体外重组萌发试验来测试改变PI4P水平是否会驱动逆行ERGIC囊泡的形成。在我们的第二个子目标中，我们将验证非常规陷阱YKT6介导了逆行小泡与NE的融合。我们还将使用我们发现的容易检测到的NE表型来识别介导NE融合的分子。总而言之，这是一种系统的方法来定义调节囊泡运输到NE的分子机制。*AIM 2研究了干扰到NE的逆行运输如何影响核孔的通透性。我们发现，外周核孔蛋白预先组装在细胞质的膜泡中，依赖于VAPB最终输送到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。我们将使用糖皮质激素受体的进口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进入细胞核，进口速度反映了它通过核孔的过程。我们还将使用TDP-43和C9orf72作为依赖于核质运输的蛋白质的例子。两者在受损时都与过早的神经元丢失有关。我们预计，核孔缺陷将导致TDP-43的胞质重新定位和核功能的丧失。对于C9orf72，G4C2重复序列的扩展导致二肽重复序列的翻译。我们假设，这些二肽的重复表达通过直接堵塞核孔来扰乱核质运输。

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.