Exchange of macromolecules between the cytoplasm and the nucleus is mediated by dedicated transport channels in the nuclear envelope (NE) called nuclear pore complexes (NPCs). NPCs are embedded within NE at fusion sites between the inner and outer nuclear membrane. They are among the largest macro­ molecular assemblies in eukaryotic cells with an estimated mass of >50 MD (Hetzer et al., 2005; Tran, and Wente, 2006). The structural organization of NPC appears to be conserved in evolution and has been delineated in various systems using EM (Unwin, and Milligan, 1982; Hinshaw et al., 1992; Yang et al., 1998; Beck et al., 2004; Beck et al., 2007). However, no de­ tailed molecular structure of NPC is currently available. A re­ cent study combined a comprehensive proteomic analysis with a diverse set of biophysical data and computational modeling to propose a three­dimensional map of all Saccharomyces cerevisiae nuclear pore proteins (nucleoporins or Nups; Alber et al., 2007a,b). In addition, the first nucleoporin crystal structures have now been solved (Hodel et al., 2002; Berke et al., 2004; Weirich et al., 2004; Hsia et al., 2007; Jeudy, and Schwartz, 2007; Melcak et al., 2007; Schrader et al., 2008), leading to new insight into NPC structure and organization (Hsia et al., 2007; Brohawn et al., 2008). Together, these studies have demon­ strated that NPCs are constructed from 500 polypeptides, but because of their highly symmetrical organization, NPCs consist of only 30 distinct nucleoporins, all present in 8, 16, or 32 copies per NPC (Rout et al., 2000; Cronshaw et al., 2002; Alber et al., 2007a,b). Despite our detailed knowledge of the protein composition of NPCs, little is known about the pathways that lead to NPC assembly, and it remains poorly understood how NPC biosynthesis is spatially or temporally coordinated. In higher eukaryotes that undergo an open mitosis, two NPC assembly pathways can be distinguished. The first pathway occurs upon completion of mitosis, when nucleoporin subcomplexes and membrane vesicles are recruited to chromatin during NE refor­ mation (Rabut et al., 2004; Antonin et al., 2008; Dultz et al., 2008). In vitro studies using an NE assembly assay in Xenopus laevis egg extracts revealed discrete NPC assembly steps that are initiated by an early recruitment of the Nup107­160 complex (Belgareh et al., 2001; Harel et al., 2003; Walther et al., 2003) to chromatin by the DNA­interacting nucleoporin ELYS (embry­ onic large molecule derived from yolk sac)/Mel28 (Rasala et al., 2006; Franz et al., 2007; Gillespie et al., 2007). This is followed The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs. Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

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@inproceedings{Onischenko2009Jcb_2008100304, title={Jcb_200810030 475..492}, author={Evgeny A Onischenko and Leslie H . Stanton and Alexis Spain Madrid and Thomas Kieselbach and Karsten Weis}, year={2009} }