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Abstract

Nuclear pore complexes (NPCs) are large macromolecular assemblies that act as gatekeepers for nucleocytoplasmic exchange. They also restrict access to the nucleus by forming a permeability barrier. NPCs are composed of approximately 30 proteins called as nucleoporins (nups). These can be categorized into ones forming the structural skeleton of the pore and others forming the central channel thus imparting functional properties to the pore. During open mitosis in metazoans the nuclear envelope and the NPC breaks down and at the end of mitosis this process is reversed wherein the nucleoporins assemble in an ordered and regulated fashion to form the intact NPC. We are interested in functionally characterizing the role of one of the structural complexes, the Nup93 complex in NPC assembly. This complex in vertebrates is composed of nucleoporins Nup93, Nup205, Nup188, Nup53 and Nup155. It is known that Nup93 forms two distinct complexes: Nup93-188 and Nup93-205. When depleted individually these complexes are not essential for NPC assembly. The objective of this study was to know what happens when we deplete both Nup93 containing complexes together, which we did by immunodepleting all three proteins from Xenopus laevis egg extracts using antibody against Nup93. This leads to a block in the NE and NPC assembly. This phenotype observed could either be a direct consequence of absence of Nup93 or resulting from co depletion of Nup188 and Nup205. To distinguish these two scenarios we did add back experiments with recombinant Nup93 and concluded that rescue in the block is due to Nup93 alone and not due to the two sub complexes implying that Nup188 and Nup205 together dispensable for NPC assembly. To know which region of Nup93 contributes to the NPC assembly and function we added back different fragments of Nup93. Surprisingly, the C terminus of Nup93 alone could rescue the phenotype. (Closed nuclear envelope and formation of structural backbone of NPC) As its known that p62 complex lining the central channel imparts the functional properties to the pore and it interacts with the N terminus of Nup93, we checked this by nuclear import and size exclusion assays. Nuclei where only the C terminus of Nup93 was present were not competent for import and lost their permeability barrier. In contrast nuclei where nup93 full length was added back were transport competent.