Dissecting the molecular machinery of nuclear transport

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DESCRIPTION (provided by applicant): ABSTRACT Nuclear pore complexes (NPCs) are the sole mediators of exchange across the nuclear envelope (NE) between the nuclear and cytoplasmic compartments. Nucleocytoplasmic transport depends on the interplay between transport cargoes, their cognate soluble transport factors (many termed Kaps), and NPCs. We have taken a comprehensive approach to defining the functional architecture of the NPC in the model eukaryote Saccharomyces (yeast). We identified all the yeast NPC proteins (Nups) and plotted their disposition in the NPC; this work allowed us to propose a new "virtual gating" mechanism for nuclear transport. We also assigned fold types to all the Nups and systematically isolated Nup subcomplexes to determine the network of interactions they make. We then used this information to compute a 3D map of the NPC architecture, sufficient to resolve the molecular organization of the entire NPC. Our work exposed a simple modularity in the architecture of the NPC; moreover, similarities between structures in coated vesicles and those in the NPC suggest their common evolutionary origin in a progenitor "protocoatomer". Our goal is now to produce high resolution dynamic maps of the NPC. First, we will use additional "low-fruit, high-payoff' immunopurification and immunolocalization experiments to rapidly improve our NPC map, to the point at which we can discern the shapes of the Nups in it. We will then study recombinant Nups and Nup complexes using electron microscopy and crosslinking, to reveal fine details on how the folds, domains, and proteins are organized within the Nups and their complexes. Next, we will reconstitute key reactions of nucleocytoplasmic transport in vitro, and test possible mechanistic models in vivo, in order to reconstruct the movements Kaps and their cargos make on crossing the NPC. We will finally synergistically decode this information and convert it into dynamic, 3D representations of the NPC and nuclear transport, ideally at atomic resolution, thereby allowing us to understand the origin, assembly and mechanism of the NPC at the most fundamental level. LAY SUMMARY We are studying the tiny machines that shuttle materials to the DNA in living cells. These machines, called "nuclear pore complexes", allow the DNA to send its instructions to the rest of the cell, and so help regulate how a cell lives, develops, and stops itself from making the kinds of mistakes seen in cancer cells. We wish to understand how these machines work and how they arose in the early evolution of life.