Jcb_200810016 387..396

The nuclear pore complex (NPC) is a supramolecular assembly that is inserted in the nuclear envelope (NE) and mediates nucleocytoplasmic transport (Fahrenkrog and Aebi, 2003; Tran and Wente, 2006). The NPC scaffold is characterized by distinct substructures that are arranged with an eightfold rotational sym­ metry (Stoffler et al., 1999; Beck et al., 2007). The NPC in the yeast Saccharomyces cerevisiae is 60 MD in size and consists of multiple copies of 30 different proteins called nucleoporins (Nups; Rout et al., 2000). One of the major structural components of the NPC is Nup170, which is required for the maintenance of a normal NPC structure and is implicated to have a role in NPC biogene­ sis (Aitchison et al., 1995). Nup170 and its highly related homo­ logue Nup157 were identified in a synthetic lethal screen using the nuclear pore membrane protein Pom152. Neither NUP157 nor NUP170 are essential, but deletion of both genes caused a lethal phenotype. Genetic analyses showed that a nup170∆ pom152∆ double mutant was lethal, whereas the combination nup157∆ pom152∆ allowed cell growth. Repression of NUP170 in a pom152 background induced the formation of an irregu­ larly shaped NE with massive extensions and invaginations. In addition, structures interpreted as partially assembled NPCs were described in the respective strains to be located below the double membrane (Aitchison et al., 1995). Nup170 was shown to be required for normal stoichiometry of FG Nups and to be involved in localizing specific FG Nups (e.g., Nup1 and Nup2) within the NPC (Kenna et al., 1996). Moreover, Nup170 forms a complex with Nup53 and Nup59 and also associates with the nuclear transport receptor Kap121 (Marelli et al., 1998). Nup53 itself was shown to interact with Ndc1, which is another nuclear pore membrane protein, suggesting that Nup53 could bridge between the NPC core and the pore membrane, making it a prime candidate to affect both nuclear pore membrane and NPC biogenesis in conjunction with Nup170 (Galy et al., 2003; Mansfeld et al., 2006; Hawryluk­Gara et al., 2008). Consistent with this possibility, overproduction of Nup53 disturbed NE organization (Marelli et al., 2001). Nup170, as well as Nup188, another large structural Nup, has also been implicated in the control of NPC permeability by altering the diameter for pas­ sive diffusion in nup170 cells (Shulga et al., 2000). How individual nucleoporins (Nups) perform their role in nuclear pore structure and function is largely unknown. In this study, we examined the structure of purified Nup170 to obtain clues about its function. We show that Nup170 adopts a crescent moon shape with two structurally distinct and separable domains, a -propeller N terminus and an -solenoid C terminus. To address the individual roles of each domain, we expressed these domains separately in yeast. Notably, overexpression of the Nup170 C domain was toxic in nup170 cells and caused accumulation of several Nups in cytoplasmic foci. Further experiments indicated that the C-terminal domain anchors Nup170 to nuclear pores, whereas the N-terminal domain functions to recruit or retain a subset of Nups, including Nup159, Nup188, and Pom34, at nuclear pores. We conclude that Nup170 performs its role as a structural adapter between cytoplasmically oriented Nups and the nuclear pore membrane. Two structurally distinct domains of the nucleoporin Nup170 cooperate to tether a subset of nucleoporins to nuclear pores