Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references (p. 127-142).The Nuclear Pore Complex (NPC) is a ~50 MDa protein complex that forms the sole conduit for macromolecular transport across the nuclear envelope. It assembles from ~30 proteins, termed nucleoporins or nups, symmetrically arranged about a central 8-fold axis. Some nucleoporins also contribute to other structures or perform diverse other functions. A subset forms a stably-associated core scaffold for the NPC, organized into two large subcomplexes, the Y-complex and the Nic96-complex. Studies using electron microscopy and X-ray crystallography have begun to elucidate the architecture of the NPC scaffold. To better understand the NPC and related proteins, two studies were performed: 1. The crystal structure of the C-terminal a-helical domain of Nup133 in complex with the C terminus of Nup107 revealed (with a prior structure of the N-terminal β-propeller domain of Nup133) the complete structure of Nup133 and its connection to the Y-complex. This contributes to a nearly complete molecular model of the Y-complex. Nup107-Nup133 forms the distal half of the stalk of the Y-complex. Sequence similarity between Nup133 and another nucleoporin, Nup170, was detected, and structural similarity proven by solving the structure of the a-helical domain of Nup170. Nup170 is a member of the Nic96-complex. Similarity between Nup133 and Nup170 suggests that the Y-complex and the Nic96-complex employ the same architectural principles. 2. Four nucleoporins contain an a-helical domain structurally related to the COPII coat protein Sec31. This domain is called the ancestral coatomer element 1 (ACE1). Sec13-Sec31 and Nup84-Nup145C-Sec13 complexes form analogous edge elements for the COPII coat and the NPC. A sequence-based search for other ACE1s identified the COPII accessory protein Sec16. Sec16 and Sec13 were shown to form a 2:2 heterotetramer. A crystal structure of Sec13-Sec16 revealed similarities to the Sec13-Sec31 edge element. Together with other structural and in vivo data, this result suggests that Sec13-Sec16 is a template for the Sec13-Sec31 coat. These studies demonstrate that duplication of multiple classes of architectural proteins occurred in the evolution of the NPC and COPII coat, and support the hypothesis that these systems evolved from a common, ancestral membrane-coating complex.by James Richardson Ross Whittle.Ph.D
