Identity and role of integral membrane proteins of Nuclear Envelope Precursor Vesicles in Membrane Fusion and Nuclear Pore Assembly. (360G-Wellcome-086169_Z_08_Z)

Two distinct Nuclear Envelope Precursor (NEP) vesicles in Xenopus eggs, NEP-A and NEP-B, are essential for Nuclear Envelope (NE) assembly. Recently, we showed that fusion between NEP-A and NEP-B initiates the formation of Nuclear Pore Complexes (NPCs). This might be because essential components of NPCs are segregated between each NEP and can only interact following vesicle fusion. Alternatively, remodelling of membranes during fusion might topologically favour NPC assembly. The proposed investig ations will test these hypothesise. Our first objective is to use a proteomic approach (MudPIT) to identify NEP-A and NEP-B specific proteins. Our second objective is to use a rational bioinformatics approach to identify those proteins in NEP-A that could be involved in NPC assembly (we already know which NEP-B proteins are involved in NPC assembly). Our third objective is to use immunogold E.M, live confocal imaging and protein-protein interaction assays to further refine the list of NEP-A prot eins likely to be involved in NPC assembly (because they are located within NPC assembly intermediates and/or interact with NPC proteins). Our final objective is to use functional assays to determine whether, within a final list of candidate proteins, some or all are necessary for NPC assembly.