Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export

Abstract

Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.

FIG. 1

Comparison of NTF2 and NXT1 proteins from diverse species. (A) The primary structure of NTF2 proteins from human (hNTF2), fly (dmNTF2), worm (ceNTF2), yeast (scNTF2), and Arabidopsis (atNTF2) were aligned with NXT1 proteins from human (hNXT1), mouse (mNXT1), fly (dmNXT1), and worm (ceNXT1). Human NXT1 and NTF2 are 26% identical over 118 amino acids. The invariant glutamate in NTF2 (Glu42 in hNTF2) that is required for binding Ran-GDP is indicated (∗). Notably, NXT1 contains an invariant asparagine at this position (Asn48 in hNXT1). Mouse NXT1 also shows 30% identity to amino acids 14 to 135 of RasGAP SH3-binding protein (33) (accession no. AF051311), though the biological significance of this relationship is unknown. (B) Phylogeny of NTF2/NXT1 generated from the sequence alignment (9). Mouse NXT1 was omitted from the phylogeny because it is 99% identical to its human orthologue. Phylogenies of the same sequences using the neighbor-joining and maximum parsimony methods yielded identical topologies (data not shown). Mtr2p was not included in the phylogeny because its sequence is not significantly similar to that of NXT1, despite the fact that the proteins have functional similarities (21). In a FASTA search of the SwissProt database, the alignment of yeast Mtr2p and mouse NXT1 protein sequences has an expectation value of 340. In contrast, the alignment of mouse NXT1 and yeast NTF2 has an expectation value of 10⁻⁴. NXT1 is also not related to the Ran-GTP-binding protein Mog1p, a recently described nuclear import factor that interacts genetically with NTF2 in the budding yeast S. cerevisiae (32).

FIG. 2

NXT1 is functionally distinct from NTF2. (A) Ribbon diagram of the NTF2 monomer from the crystal structure (4) (Protein Data Bank no. 1oun, chain A) compared to the structure of mouse NXT1 generated with the Modeller program. The invariant glutamate in the Ran-binding domain of NTF2 is replaced with an invariant asparagine in NXT1. (B) BPY4(YCplac33-NTF2) was transformed with pRS315 (vector), pRS315-NTF2 (NTF2), or pRS315-ntf2 E40N (E40N). Individual colonies were restreaked on 5-fluoro-orotic acid-containing medium. The E40N mutation was lethal, as there was no growth in this strain upon counterselection of the wild-type copy. (C) BPY4(pRS315-MET3-NTF2) was transformed with pMSS32 (vector), YEp24-NTF2 (NTF2), or pMSS32-NXT1 (NXT1). Individual colonies were restreaked on medium containing 5 mM methionine. Upon repression of NTF2 expression, strains containing a plasmid copy of NTF2 were viable, but those containing vector alone or a plasmid copy of NXT1 failed to grow.

FIG. 3

NTF2 and NXT1 interact with different nucleotide-bound forms of Ran. Recombinant NTF2 and NXT1 were immobilized in microtiter wells, to which Ran preloaded with [³H]GDP-Ran (A) or [γ-³²P]GTP-Ran (B) was added. NTF2 preferentially bound GDP-Ran (A), whereas NXT1 preferentially bound GTP-Ran (B). (C and D) Binding assays were also carried out with mixtures of [α-³²P]GDP-Ran and [α-³²P]GTP-Ran. When presented with mixed nucleotide forms of Ran, NTF2 and NXT1 specifically bound to GDP-Ran and GTP-Ran, respectively. (E) The affinity of Ran binding was measured by performing binding reactions with increasing concentrations of Ran preloaded with [γ-³²P]GTP.

FIG. 4

NXT1 does not affect RanGAP-stimulated GTP hydrolysis of Ran. (A) Recombinant Ran preloaded with [α-³²P]GTP was incubated with recombinant RanGAP for 10 min with (●) or without (■) NXT1. The reactions were terminated by the addition of SDS and analyzed by thin-layer chromatography. (B) Similar GAP assays were carried out with Ran preloaded with [α-³²P]GTP and incubated with RanGAP (1 nM) in the presence of buffer alone, NXT1 (7 μM), or RanBP1 (7 μM). Samples were taken at 0, 4, 8, 16, and 48 min and analyzed by thin-layer chromatography.

FIG. 5

Subcellular distribution of NXT1. (A) Indirect immunofluorescence microscopy of HeLa cells stably transfected with pFlag-NXT1, showing that NXT1 is a nuclear protein. NXT1 also localizes to the nuclear envelope. After digitonin extraction, NXT1 is released from the nucleoplasm but remains associated with the nuclear envelope (A, lower row). Nuclear Ran is released from the nucleus under these conditions. (B) NXT1 colocalizes with the NPC. Flag-NXT1 cells were labeled with α-Flag antibodies (red) and NPC-specific antibodies (green). The merged image reveals the coincident localization of NXT1 and NPCs (yellow).

FIG. 6

NXT1 shuttles between the nucleus and cytoplasm in living cells. The Flag-NXT1 cell line was fused with a cell line stably expressing constitutively nuclear GFP-NLS. Heterokaryons were analyzed by immunofluorescence to localize Flag-NXT1 (red) and GFP-NLS (green). In the heterokaryon shown, Flag-NXT1 protein was exported from a single donor nucleus and imported into three acceptor nuclei containing GFP-NLS. NXT1 shuttling was observed in multiple heterokaryons from three separate experiments.

FIG. 7

NXT1 is a nuclear export factor. (A) NXT1 stimulates NES-dependent nuclear export in digitonin-permeabilized cells. Export reactions were performed with HeLa cell cytosol in the absence and presence of recombinant NXT1 (100 μg/ml). The postexport nuclear fluorescence was measured in ∼50 randomly selected nuclei by digital fluorescence microscopy and plotted as the mean nuclear fluorescence (±SD) (16). We also found NXT1 could stimulate PKI export in the absence of cytosol (data not shown). (B) Dose-response of NXT1-stimulated nuclear protein export. Export reactions were performed at a subsaturating concentration of cytosol and increasing concentrations of NXT1. We note that NTF2 assayed under similar reaction conditions also stimulates nuclear export, though to a lesser extent (data not shown). The nuclear export stimulation by NTF2 is probably linked to its ability to increase the nuclear concentration of Ran in permeabilized cell nuclei. (C) LMB blocks NXT1-stimulated nuclear protein export. The stimulation of nuclear protein export by NXT1 is blocked in the presence of LMB, a specific inhibitor of Crm1 (51).