Here we show for the first time that histones H2A and H2B both contain distinct NLSs in their amino-terminal tails. We demonstrate that the import factor Kap114p forms a cytoplasmic complex with the H2A/H2B heterodimer, via direct interactions with both NLSs. H2A and H2B also interact with other Kaps, including Kap121p, Kap95p, and Kap123p. Experiments in vivo suggest that each of these Kaps participates in H2A/H2B import and we propose that the import of abundant, essential proteins such as histones is mediated by a network of Kaps.
Histones H2A and H2B are imported into the nucleus as a heterodimer. We have demonstrated that both H2A and H2B have a functional NLS in their amino termini. Amino acids 21–33 of H2B represents a “minimal” NLS domain, which correlates with the NLS previously reported by Moreland et al. 1987
in fixed cells. H2B1-52
was more nuclear, suggesting that the amino acids flanking the minimal domain are also important. We identified an NLS domain in H2A consisting of amino acids 1–46, although residues 24–46 gave some nuclear accumulation. In vitro binding experiments showed that amino acids H2A1-46
were sufficient to confer Kap114p binding. In vivo, it remains to be determined whether one or both NLSs in the H2A/H2B heterodimer simultaneously bind to Kap114p. Comparison of these two amino-terminal NLS sequences does not reveal any obvious similarities. Since the amino-terminal domains of histones have been shown to be substrates for several posttranslational modifications (Strahl and Allis 2000
), it is possible that these modifications may also play a role in NLS recognition. Similarly, posttranslational modifications of the transcription factor Pho4p regulate its interaction with its import Kap (Kaffman et al. 1998
Kap114p also imports TBP into the nucleus, and this Kap may have several cargoes (Pemberton et al. 1999
). It is intriguing that the same Kap imports the H2A/H2B histone dimer and TBP. These proteins are fundamental for all aspects of gene regulation, but are generally perceived to have opposing effects. Sequence comparison of the NLSs of H2A and H2B with TBP does not reveal any obvious similarities at the amino acid level. Kap114p interacts directly with the NLSs of H2A, H2B, and with TBP (Pemberton et al. 1999
). In vivo, we do not know whether the Kap114p-TBP and Kap114p-H2A/H2B complexes are assembled independently or on the same Kap. How three apparently different NLSs could interact with the same domain on Kap114p is unclear, and structural determination of Kap114-cargo interactions should elucidate the mechanism of binding.
The NLS of H2B is rich in basic amino acids, suggesting it may represent a “classical” NLS (Moreland et al. 1987
). However, we have no evidence of Kapα binding to either the H2A or H2B NLS by PrA coimmunoprecipitation or by localization of the reporters in the Kapα ts strain, srp1-31
. This data correlates with experiments in mammalian cells, where H2B import was not blocked by addition of excess SV40 NLS peptide in a permeabilized cell system (Langer 2000
), suggesting that histones are imported by an α-independent pathway.
The biochemical coisolation of endogenous Kap114p, Kap121p, and Kap123p with H2A-PrA and H2B-PrA suggested that these Kaps mediate the import of H2A and H2B. Kap114p may play a more major role in H2A/H2B import, suggested by the fact that Kap114p was the predominantly isolated Kap. Kap114p is less abundant than Kap121p, Kap123p, and Kap95p (Morehouse et al. 1999
; Pemberton et al. 1999
). It is intriguing that when H2A-PrA, H2B-PrA, and TBP-PrA cytosolic complexes were purified, Kap121p and Kap123p were only readily detectable in the absence of Kap114p, and these Kaps were eluted from histones with lower concentrations of MgCl2
. This suggests that these cargoes preferentially bind Kap114p before Kap121p and Kap123p.
To determine the role of these Kaps in vivo, we investigated the localization of H2A NLS-GFP and H2B NLS-GFP reporters in the corresponding Kap mutant strains. The H2A and H2B NLS-GFP reporters were mislocalized in strains with mutations in kap114, kap121, kap123, and kap95. Quantitation of fluorescence showed a significant decrease in the N:C ratios between wild-type and these mutant strains. In addition, an additive effect was measured in the Δkap114/kap121 ts mutant, with significantly more cytoplasmic mislocalization of both reporters than was detected in either single mutant. This demonstrates that both these Kaps function in histone import in vivo. We do not know why the effect was greater in the kap121ts strain than the Δkap114 strain, as H2A and H2B appear to preferentially bind Kap114p. It is possible that there are other pleiotropic defects in nuclear transport in this temperature-sensitive strain.
These experiments also suggested that Kap95p functioned in H2A import and to a lesser extent H2B import. We did not detect Kap95p by MS in our immunoprecipitation experiments using endogenous proteins. We could, however, show binding of Kap95p to the H2A and H2B NLSs using recombinant proteins. In addition, published studies using recombinant mammalian proteins in ELISAs suggest that Kapβ1 (human Kap95) may directly bind H2A/H2B in vitro (Johnson-Saliba et al. 2000
). We do not know whether additional Kaps can import histones H2A and H2B. Surprisingly, we did detect a small decrease in the N:C ratio of our “control” Kap, Kap108, that was not obvious from the fluorescent images. This decrease was less than that observed for kap114
deletions. We cannot rule out the possibility that Kap108p plays a very minor role in import, or that this a pleiotropic defect of the mutant strain. Together, our in vivo data, both the isolation of endogenous protein complexes and the localization of NLS reporters in mutant strains, suggest a major role for Kap114p and Kap121p, and Kap123p and Kap95p in H2A and H2B import.
Our in vitro binding studies using recombinant NLSs and Kaps expressed in reticulocyte lysate or purified from bacteria confirmed that the same NLSs are recognized by Kap114p, Kap121p, Kap95p, and Kap123p. We were able to show that these interactions were specific as they were blocked by preincubation of the Kaps with RanGTP. Future experiments should determine whether additional factors stimulate the RanGTP-mediated dissociation of histones from Kaps, as was observed with Kap114p and TBP (Pemberton et al. 1999
). Although both H2A and H2B interact similarly with Kap114p, our results suggest that there maybe some subtle differences between the interactions of H2A and H2B with the other Kaps. We have evidence that more Kap121p and Kap123p are isolated with H2B. In fact, Kap123p was detected in the H2A-PrA coisolation by MS, but not detected by Western blotting, suggesting that it was present at lower levels than observed with H2B-PrA. In addition, we observed a more dramatic decrease in the N:C ratio of the H2B NLS-GFP, than of H2A NLS-GFP, in the kap121ts
strain. In contrast, evidence that Kap95p appears to function preferentially with H2A comes from both the in vivo localization and in vitro binding experiments. It is interesting that these Kaps, which have low apparent sequence homology, bound the same NLSs in H2A and H2B.
In addition to the import of TBP, which is mediated by the same subgroup of Kaps discussed here (Pemberton et al. 1999
), the only other published examples of multiple Kaps importing a single cargo are in the Kap123p- and Kap121p-mediated import of yeast ribosomal proteins. There, Kap123p mediates the primary route into the nucleus (Rout et al. 1997
). Multiple Kaps have also been shown to import mammalian ribosomal proteins into the nucleus in vitro (Jakel and Gorlich 1998
). However, in these experiments, it is not possible to compare the specific in vivo requirement for, and the contribution by, each Kap. Further examples of networks of import Kaps need to be characterized to determine their importance and hierarchy in vivo. We suggest that the import of essential proteins by several Kaps will represent a broadly relevant model.
It is probable that Nap1p interacts with the Kap114p-histone complex via one of the histones. The majority of cytosolic Nap1p is not complexed with Kap114p (Pemberton, L.F., unpublished data) and may be interacting with a pool of free histone monomers or dimers. One model is that Nap1p binds to histones as they are synthesized and, upon the interaction of Kap114p with the histone dimer, some fraction of Nap1p remains bound to the Kap114p-histone complex. This may be the route of Nap1p into the nucleus, where it carries out chromatin assembly functions. Immunofluorescence studies have demonstrated that the majority of Nap1p remains in the cytoplasm, and distinct cytoplasmic functions in mitosis have been determined (Kellogg et al. 1995
; Kellogg and Murray 1995
; Ito et al. 1996
). We have not detected any Nap1p in a complex with Kap123-PrA or Kap121-PrA in the presence of Kap114p (Pemberton, L.F., unpublished data). This may be due to the fact that there is little histone bound to these Kaps in the presence of Kap114p, or that Nap1p may be functionally linked to the Kap114p-mediated import pathway. It is possible that Nap1p may help regulate the interaction between histones and Kap114p.
We propose a model where histone H2A/H2B is synthesized and heterodimerizes in the cytoplasm, and is bound by the chaperone Nap1p. The majority of this complex is imported into the nucleus by Kap114p. However, a fraction of H2A/H2B is imported by Kap121p or Kap123p, preferentially binding via H2B, and some is imported by Kap95p, binding via H2A. Future experiments will determine what factors regulate which Kap pathway is used, and whether different pathways are used under different conditions.
H2A and H2B are synthesized in S phase and must rapidly enter the nucleus as DNA is replicated. The production and import of H2A and H2B needs to also be coordinated with that of H3 and H4. Because of the importance of having the correct amounts of histones in the nucleus, it is not surprising that the import of these proteins is mediated by several independent transport factors. We propose that all essential proteins may have more than one route into the nucleus, explaining why the Kap family members are functionally overlapping.