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Abstract

The determining property of eukaryotes is the compartmentalization of genetic information inside the cell nucleus. This compartmentalization by the nuclear envelope (NE) enables the eukaryotic cell to separate transcription and translation in space and time. The NE is composed of two membranes, the outer (ONM) and inner nuclear membrane (INM). The ONM is an extension of the endoplasmic reticulum whereas the INM contacts chromatin and the nuclear lamina, and is connected to the ONM via the pore membrane, the region where nuclear pore complexes (NPCs) are localized. Although, these membranes are continuous with each other, the INM contains a distinct set of proteins and the mechanism of INM protein targeting is not yet completely understood. NPCs are the gateway to the nucleus; they are embedded on the NE where INM and ONM are fused. They are about 60 MDa in vertebrates, and, are comprised of around 30 distinct proteins called nucleoporins or nups many which nucleoporins form subcomplexes that act as building blocks for NPC assembly. This thesis aims to characterize the Nup188-Nup93 complex during NPC assembly and function. One of the evolutionarily conserved subcomplexes is the Nup93 complex. It is a major structural component of the NPC which is thought to be positioned at the region where the NPC and nuclear membranes interact. In Xenopus, the core of the Nup93 complex has been understood to be formed by three proteins: Nup205, Nup188 and Nup93. Using biochemical and cell biological assays we show that Nup93 is not part of a single complex but is in fact a component of two distinct subcomplexes, Nup188-Nup93 and Nup205-Nup93. Using in vitro nuclear assembly reactions, we find that neither Nup188-Nup93 nor Nup205-Nup93 is required for NPC or NE formation at the end of mitosis. However, nuclei lacking Nup188-Nup93 increased dramatically in size compared to normal nuclei. Our analysis of Nup188-Nup93 depleted nuclei demonstrates that the DNA content and nucleocytoplasmic transport of soluble proteins, mRNA and small molecules are similar to normal nuclei. We show, through a novel assay, that the enlarged nuclear phenotype observed in the absence of Nup188-Nup93 correlates with an increase in translocation of integral membrane proteins to the INM through NPCs. The phenotype is rescued with recombinant Nup188-Nup93 confirming the specificity of the depletion effect. This work establishes the idea that Nup188-Nup93 limits the passage of membrane proteins through the NPC and is thus crucial for the homeostasis of nuclear membranes. In addition, our results also strengthen the view that integral membrane proteins of INM reach to the interior of nuclei by passage from the ONM through the NPC in the plane of membrane.