Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p, Nup59p, and Nup170p

Abstract

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.

Vertebrate Nup53 Interacts with the Nuclear Lamina and Is Required for the Assembly of a Nup93-containing Complex

“…How Nup59p, Nup157p, and Nup192p fit into this developing model is less clear. Proposals must consider the fact that Nup59p and Nup157p are likely products of gene duplications that also gave rise to Nup53p and Nup170p, respectively (Aitchison et al, 1995b;Marelli et al, 1998). Thus, although these pairs of related proteins are not functionally identical, it is possible that they are structurally interchangeable within the repetitive subunits of the NPC, creating a mosaic of structurally similar subcomplexes composed of different combinations of these related nups.…”

“…Genetic and biochemical analyses have established that yeast Nup53p is part of a network of interacting nups that includes Nup170p, Nup59p, Nic96p, Nup157p, and Nup192p (Aitchison et al, 1995b;Marelli et al, 1998;Kosova et al, 1999;Fahrenkrog et al, 2000;Lusk et al, 2002;Makhnevych et al, 2003). A model for how these nups are organized within the NPC is beginning to emerge.…”

“…Amino acid sequence analysis has previously identified potential vertebrate orthologues of the S. cerevisiae nucleoporin Nup53p (Marelli et al, 1998). These multiple ORFs encode proteins with molecular masses between 35 and 36 kDa and exhibit similarity to Nup53p in four distinct regions, each separated by an insertion in the larger yeast version (unpublished data).…”

“…In yeast, Nup53p is part of a defined subcomplex of nups (Marelli et al, 1998;Fahrenkrog et al, 2000;Lusk et al, 2002) and Nup53p has been suggested to interact directly with the NE membrane (Marelli et al, 2001). We have examined the association of vertebrate Nup53 with NEs using differential extraction procedures and Western blotting.…”

“…For detection of Nup53, antibodies were first affinity purified as described (Harlow and Lane, 1988) with modifications (Marelli et al, 1998). After incubation with primary antibodies, coverslips were washed three times with PBS-T over a period of 45 min.…”