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）囊泡不直接进入高尔基体，而是相互融合形成ER-高尔基体中间区室（ERGIC）。要到达高尔基山脉，需要从ERGIC进行第二步贩运。同样，来自高尔基的逆行货物在通往ER的途中也要经过ERGIC。我们发现VAPB （VAMP-Associated Protein B）通过ERGIC调控逆行交通流。此外，我们发现核膜（NE）蛋白也通过ERGIC转运，VAPB介导其最终递送到NE。这一发现提供了一个独特的机会：(1)确定介导向NE逆行运输的分子机制，(2)定义破坏逆行运输对核质运输的细胞后果。

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.