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Until very recently, the vertebrate protein Npap60/Nup50 was thought merely to be a component of the nuclear pore complex (NPC). This conclusion was based on the observations that Npap60/Nup50 localizes at the NPC by immunofluorescence and electron microscopy and also contains FG (Phe-Gly) repeats, a motif commonly found in nucleoporins but not in proteins located elsewhere. However, far from being a fixed structural component of the NPC, it now appears as though Npap60 can shuttle from one side of the NPC to the other. Most significantly, a recent paper shows that Npap60 enhances the nuclear import of a cargo possessing a basic nuclear localization sequence by associating directly with the import cargo–carrier complex and (presumably) moving through the NPC with it. Several NPC proteins have now been shown to be mobile in the NPC, and this new report might indicate that these ‘mobile’ nucleoporins play a more active role in the nuclear transport of cargo than was previously appreciated. [Copyright &y& Elsevier]

Abstract: In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-β. Crystallization studies by others have indicated that nuclear carriers such as importin-β bind to phenylalanine–glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-β identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-β. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-β binding to those repeats. In addition to these expected binding sequences for importin-β, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-β, indicating that additional Nup sequences may form binding sites for importin-β. [Copyright &y& Elsevier]

The nuclear import of classical nuclear localization signal-containing proteins depends on importin-α transport receptors. In budding yeast there is a single importin-α gene and in higher eukaryotes there are multiple importin-α-like genes, but in fission yeast there are two: the previously characterized cut15 and the more recently identified imp1. Like other importin-α family members, Imp1p supports nuclear protein import in vitro. In contrast to cut15, imp1 is not essential for viability, but imp1Δ mutant cells exhibit a telophase delay and mild temperature-sensitive lethality. Differences in the cellular functions that depend on Imp1p and Cut15p indicate that they each have unique physiological roles. They also have common roles because the imp1Δ and the cut15-85 temperature-sensitive mutations are synthetically lethal; overexpression of cut15 partially suppresses the temperature sensitivity, but not the mitotic delay in imp1Δ cells; and overexpression of imp1 partially suppresses the mitotic defect in cut15-85 cells but not the loss of viability. Both Imp1p and Cut15p are required for the efficient nuclear import of both an SV40 nuclear localization signal-containing reporter protein and the Pap1p component of the stress response MAP kinase pathway. Imp1p and Cut15p are essential for efficient nuclear protein import in S. pombe. [ABSTRACT FROM AUTHOR]

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC. [Copyright &y& Elsevier]