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Nuclear pores are established at fusion points of the outer and the inner nuclear membrane and are stabilized by the proteins of the nuclear pore complex (NPC). These huge protein assemblies form the selective gate, which allows a bidirectional transport, and also the scaffold maintaining the highly bent pore membrane structure. Although the last years have seen advances in the determination of the NPC structure and its components, the necessary factors and processes for the formation of the nuclear pore are still ill defined. My work describes the involvement of the nucleoporin Nup53 in the formation of the nuclear pore and the assembly of NPCs. Nup53 is part of the Nup93 complex, which is one of two subcomplexes that form the structural backbone of NPCs. By the use of in vitro systems and the application of recombinant proteins, I was able to demonstrate a direct membrane binding of Nup53, a property that is crucial for NPC assembly. The vertebrate protein comprises two membrane-binding sites, of which the C-terminal domain has membrane-deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact nuclear envelope during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly. This investigation illustrates the importance of Nup53’s membrane interaction for the formation of NPCs and it identifies mechanistic differences in the formation of NPCs depending on the cell cycle stage.