The goal of our research is to obtain an understanding of the mechanisms of nucleocytoplasmic transport and co-/post translational translocation. Nucleocytoplasmic transport across the Nuclear Pore Complex is instrumental in the control of gene expression. Co- and post translational translocation are mediated by the ribosome- Sec61p and Sec complexes and play crucial roles in the targeting of secretory and membrane proteins. This proposal describes ongoing studies which utilize cryo-electron microscopy and 3D image processing to determine functionally relevant structures of these machines. (a)Translocation mediated by the NPC-transporter. Our 3D studies suggest that the NPC-transporter is a cylindrical channel that resides within a flexible spoke-ring complex. The """"""""conformational signaling"""""""" hypothesis proposes that spoke plasticity may be utilized by lumenal effectors to down-regulate transport. Proposed 3D studies will utilize improvements in specimen preparation, data collection and """"""""cylindrical unbending"""""""" to markedly improve resolution on NPCs containing distinct configurations of the spoke-ring complex and to visualize the transporter in closed and open forms. This will allow an evaluation of the """"""""macromolecular lock"""""""" hypothesis, which postulates that an asynchronous dilation of opposing transporter gates occurs during translocation. These studies will encompass both vertebrate and yeast NPCs. (b) Co- and post-translational translocation. Our initial studies have shown that the Sec61p heterotrimer forms a highly conserved ring-like oligomer (with p1 symmetry) in both the co-and post-translocational complexes. This oligomer has a central ~20A diameter pore and may function as a protein conducting channel. We propose to solve 3D structures of the ribosome-Sec61p assembly and the yeast Sec complex by single particle methods. In addition, we have initiated 2D crystallization studies with the Sec61p complex from cow/yeast and the homologous SecYG complex from B. subtilus. These studies have yielded large paracrystalline tubes comprised of Sec61p heterotrimeric subunits from cow and B. subtilus, that are membrane free. Further improvements may provide a helically-derived 3D map of the Sec61p subunit, which in turn will be used to interpret maps obtained from the channel complexes.
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