The long term goal of this research is to obtain an understanding of the mechanism of nucleocytoplasmic transport mediated by the Nuclear Pore Complex (NPC). This process governs the translocation of macromolecule between cytoplasmic and nuclear compartments and is instrumental in the control of gene expression during cell growth and development. Initially, soluble receptors may mediate substrate docking to the NPC- transporter. Translocation across the nuclear envelope and substrate release complete the cycle. This proposal will delineate ongoing studies utilizing cryo-electron microscopy and 3D-image processing to study translocation and regulatory mechanisms of nucleocytoplasmic transport: (a) Translocation mediated by the NPC-transport and conformational regulation. Our 3D studies have shown that the NPC-transporter is a tripartite cylinder with a central channel, that resides within a conformationally flexible spoke-ring complex. Proposed 3D studies will utilize improvements in specimen preparation, data collection and a novel """"""""cylindrical unbending"""""""" approach to markedly improve resolution on NPCs containing distinct configurations of the spoke-rink complex and the transporter. In this way, structural details of the gating subunits within the central transporter will be visualized in closed and """"""""in transit"""""""" forms. This in turn, will provide insights into the """"""""macromolecular lock"""""""" hypothesis which postulates and synchronous dilation of opposing gates during substrate translocation. Moveover, the """"""""conformational pathway to globally down-regulate transport. This hypothesis will be investigated by solving 3D structures of the spoke- ring complex in distinct conformations, induced by specific isolation of intact nuclei with or without lumenal Ca+2. (b) Comparison of the structure of yeast and vertebrate NPCs and labeling studies. Our data indicates that the yeast NPC is a smaller version of the amphibian pore complex, in which the 8-fold symmetry and dimensions of the central cavity which houses the transporter are conserved. Three- dimensional structures of frozen-hydrated yNPCs prepared with and without the nuclear envelope will be obtained using the random conical tilt method. Comparison with amphibian NPCs will allow an evaluation of functionally conserved components and their potential roles in transport and regulation. Finally, epitope tagging methods will be developed to map specific nucleoporins within 3D context of the yNPC.
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