| The work in this thesis is divided into three independent chapters. The first chapter demonstrates the role of importin beta in regulating nuclear assembly. The second chapter furthers the understanding of importin beta's regulatory role through the analysis of potential targets of importin beta in nuclear assembly. Finally, the third chapter identifies and investigates the function of Xenopus gp210, a transmembrane nuclear pore protein.;Toward the end of mitosis in higher eukaryotes, nuclear assembly involves the precise targeting of nucleoporins and membranes to the surface of chromatin. This process encapsulates the genome in two nuclear membranes perforated with nuclear pore complexes, which allow transport through the nuclear envelope. Nuclear assembly is a complex process, and thus regulatory mechanisms must be in place. In studies reported here, importin beta was found to be a negative regulator of nuclear assembly. Specifically, it negatively regulates nuclear membrane fusion and, separately, the subsequent step of nuclear pore assembly. The small GTPase Ran was demonstrated to be an opposing positive regulator of importin beta in nuclear membrane fusion but, surprisingly, not in nuclear pore assembly.;To identify the regulatory targets of importin beta, proteins it interacted with were investigated. One potential target for importin beta in its block to nuclear membrane fusion is p97, a protein implicated in nuclear membrane fusion. The two proteins proved not to interact. Given the strong nature of the block to nuclear pore assembly by importin beta, it was hypothesized that importin beta would bind to and inhibit many of the nuclear pore subunit interactions involved in assembly. Instead, only a small subset of interactions between nucleoporins was perturbed. Thus, importin beta regulates the assembly of key distinct nucleoporin subunits, but may cooperate with other proteins to regulate complete nuclear pore assembly.;gp210 is a transmembrane nucleoporin, and as such, has been hypothesized to be involved in nuclear pore assembly. To understand the role of gp210 in the nuclear pore, gp210 was studied in the Xenopus egg extract nuclear reconstitution system. Xenopus gp210 was shown not to be involved in nuclear assembly, but may instead be involved in nuclear disassembly. |
