NXF1 is a well-established nuclear import cargo of Kapβ2 (Truant et al., 1999
; Bachi et al., 2000
; Lee et al., 2006
; Imasaki et al., 2007
). The karyopherin binds a PY-NLS in the N-terminal tail of hs
NXF1 (Lee et al., 2006
). Through extensive mutagenesis, qualitative and quantitative binding assays, we showed that the PY-NLS of hs
NXF1 spans residues 1–92, binds Kapβ2 with a KD
of 40 nM, and is a member of the basic and not the previously predicted hydrophobic subclass of PY-NLSs. We identified binding determinants or NLS epitopes in two distinct segments of hs
NXF1 that correspond to an N-terminal basic epitope at residues 21–30 and the R-X2-5
-P-Y motif at residues 71–75. The latter is a marginal hotspot whereby mutation of the entire five-residue motif decreased Kapβ2 binding by fivefold, whereas mutation of the former decreased affinity by threefold. The basic/hydrophobic and R-X2-5
-P-Y epitopes of previously identified PY-NLSs are connected by 3- to 11-residue-long linkers (Lee et al., 2006
). The unusually long 40-residue PY-NLS linker in hs
NXF1 significantly extends previous limits for linker length without compromising high-affinity interactions with Kapβ2.
Surprisingly, inhibition of Kapβ2 by the M9M peptide inhibitor did not mislocalize endogenous hsNXF1 in HeLa cells, suggesting that Kapβ2 is not its sole nuclear import factor. We showed that the N-terminal tail of hsNXF1 contains multiple redundant and overlapping NLSs that are recognized by Kapβ2, Impβ, Imp4, Imp11, and Impα. The five karyopherins differentially bind the same two NLS epitopes that are recognized by Kapβ2. The basic patch at residues 21–30 is used in interactions with all five karyopherins, whereas the R-X2-5-P-Y motif at residues 71–75 is used only for binding Kapβ2. The overlapping nature of the NLSs suggests that a single molecule of hsNXF1 likely binds only one karyopherin molecule at a time. Mutations of both NLS epitopes greatly diminished nuclear localization of hsNXF1, failed to rescue the nuclear accumulation of mRNA in hsNXF1 knockdown cells, and perturbed NXF1-mediated gene expression as observed by the significant decrease in reporter gene expression.
Our biochemical and biophysical characterization of the hsNXF1 NLS epitopes that bind Kapβ2, Impβ, and Impα allowed extension of these studies to other eukaryotes. The N-terminal tails of NXF1s from fission yeasts, nematodes, insects, and chordates share similar sequence/motif organizations even though they are very diverse in sequence and length. The N-terminal tails of nematode, insect, and chordate NXF1s contain N-terminal basic patches of 10–30 residues, followed by acidic patches of approximately six to eight residues and C-terminal R/K/P-X2-5-P-Φ motifs. The N-terminal tails of two fission yeast NXF1s show similar trends but lack the central acidic patches. No basic, acidic patches or R/K/P-X2-5-P-Φ motifs are present in the N-terminal tail of S. cerevisiae. The N-terminal basic patches of the NXF1s are reminiscent of the N-terminal basic NLS epitope of hsNXF1, whereas their C-terminal R/K/P-X2-5-P-Φ motifs resemble the R-X2-5-P-Y motif of the hsNXF1 PY-NLS. Functions of the central acidic patches are not known.
Individual karyopherins are highly conserved in eukaryotes, both in their sequences and cargo recognition (Enenkel et al., 1995
; Lange et al., 2008
; Suel et al., 2008
; Marfori et al., 2011
). The diverse NXF1 N-terminal tails bound similarly to human and S. cerevisiae
karyopherins, suggesting that karyopherin specificities for their NLSs are conserved from human to yeast. We found that the number of karyopherins that can mediate nuclear import of NXF1s increased steadily from fungi to nematodes and insects to chordates. Mex67p of S. cerevisiae
has no NLS and is known to be localized not to the nucleoplasm but to NPCs (Segref et al., 1997
; Katahira et al., 1999
). NXF1s from S. pombe
, C. elegans
, and human are known to be nuclear, and their N-terminal tails are important for their nuclear localization (Bear et al., 1999
; Katahira et al., 1999
; Bachi et al., 2000
; Tan et al., 2000
; Yoon et al., 2000
; Herold et al., 2001
; Wilkie et al., 2001
). Mex67p of S. pombe
bound mostly Impα, whereas the karyopherin repertoires for C. elegans
and D. melanogaster
NXF1s were expanded to include Impα, Imp11, and direct interactions with Impβ. The complexity of nuclear import is further increased in chordates, with the use of at least four karyopherins: Impβ, Kapβ2, Imp11, and Impα.
The NLS epitopes recognized by Impβ and Impα are all located within the N-terminal basic patches of the NXF1 proteins, whereas Kapβ2 recognized the R-X2-5-P-Y motifs in chordate (H. sapiens, X. tropicalis, and D. rerio) NXF1s. Of interest, the slightly divergent R/K-X2-P-I, P-X2-P-V, and R-X2-3-P-I/V motifs in S. pombe, C. elegans, and D. melanogaster, respectively, were unsuitable for Kapβ2 binding. Therefore it appears that strong R-X2-5-P-Y motifs evolved only in chordates to expand nuclear import to Kapβ2. The motif, in combination with the more primitive basic patch, produced functional basic PY-NLSs in the NXF1s of these higher eukaryotes, resulting in a total of three to five different nuclear import pathways that target NXF1s to the nuclei of human cells.
It is puzzling that the means of transporting NXF1 into the nucleus are different from S. cerevisiae to humans even though its mRNA export function is conserved. What are the advantages of increased complexity in NXF1 nuclear import or increased redundancy of NXF1 nuclear import pathways in higher eukaryotes? The simplistic suggestion that redundant nuclear import pathways are necessary to ensure correct localization of NXF1 to the nucleus for the crucial process of mRNA export is rather unsatisfactory, given that S. cerevisiae Mex67p has no NLSs and does not need to be localized to the cell nucleus at all. It is more likely that redundant NLSs in NXF1s are important to regulate mRNA export and its coupling to the upstream and downstream gene expression processes of transcription, splicing, and/or translation.
NXF1 binds mRNAs weakly, but the interaction is significantly enhanced by adaptor proteins REF and SR proteins (Hautbergue et al., 2008
). In higher eukaryotes, adaptor proteins couple mRNA export to upstream processes of capping and splicing (Izaurralde et al., 1995
; Zhou et al., 2000
; Masuda et al., 2005
; Cheng et al., 2006
). Interactions with mRNA and adaptor proteins were mapped to hs
NXF1 residues 61–118 and 1–362, respectively (Bachi et al., 2000
; Stutz et al., 2000
; Huang et al., 2003
), thus overlapping significantly with karyopherin binding. In the nucleus, the termination of NXF1 import is likely coupled to its interactions with mRNA, adaptor proteins, and upstream processes of capping and splicing. In the cytoplasm, the karyopherins that import NXF1 may contribute to its release from adaptor proteins and mRNA prior to translation. Furthermore, differential binding of Kapβ2, Impβ, Imp4, Imp11, and Impα to the N- and C-terminal NLS epitopes of hs
NXF1 may affect its interactions with various subsets of adaptor proteins, thus providing a means of regulating assembly and disassembly of diverse populations of mRNA export complexes.
Finally, the striking difference in nuclear localization of NXF1 in higher eukaryotes but not in S. cerevisiae
may reflect new and still-undetermined functions of NXF1 in the nucleus of higher eukaryotes. The increasing complexity of NXF1 nuclear import in higher eukaryotes may be correlated with similar complexity in nuclear functions of NXF1. The architecture of modular NLS epitopes within the flexible and structurally disordered N-terminal tail of NXF1 may have allowed significant evolvability to form multiple NLSs (Suel et al., 2008
). This in turn could have provided a path for NXF1 to switch from using one karyopherin to another and ultimately from one cellular process to another.