The concept of specific nuclear transport signals arose from the observation that certain proteins, larger than the passive diffusion limit (~50 kDa) for the NPC, can accumulate within the nucleus. A classic series of experiments on nucleoplasmin provided the first proof of the existence of such signals, and the first signal sequence for nuclear import was identified in the simian virus 40 (SV40) large-T antigen (53
). This sequence, PKKKRK,
and that found in nucleoplasmin, KR
), are the prototypes for the monopartite and bipartite nuclear-localization signals (NLS) now known to be present in many—probably thousands—of different proteins (Table ). A useful Web server (PSORTII) for identifying potential NLS sequences in protein sequences is available at http://psort.nibb.ac.jp:8800/
Nucleocytoplasmic signal sequences
One could imagine two likely mechanisms by which such signals might operate. Either they would bind directly to components of the NPC and be translocated through the pores like passengers on a moving walkway, or they could be recognized by soluble receptors that would carry them through the pores, like cargo carried on a truck. The development of an in vitro assay for nuclear import, using digitonin-permeabilized cells, provided the critical advance that demonstrated the second mechanism to be correct (3
). Accumulation of a fluorescently tagged protein containing an NLS into the nuclei of permeabilized cells did not occur in the absence of cytosol. Fractionation of the cytosol led to the discovery of four soluble transport factors that could, together with ATP, reconstitute nuclear import (2
). One of these factors, importinα (also called karyopherinα, Kapα, and PTAC58), was shown to bind directly to an NLS, confirming the soluble receptor-carrier hypothesis (1
The other transport factors that were isolated from cytosol using the in vitro assay were Ran, a small GTPase (also called TC4) (154
), NTF2 (also called p10 or pp15) (161
), and importinβ (also called karyopherinβ, p97, and PTAC97) (35
). The functions of each of these factors will be discussed in more detail below. For the present, it is sufficient to note that importinα itself does not interact with the NPC but instead functions as an adapter that binds to importinβ and that importinβ is the carrier that allows translocation through the pore.
These studies revolutionized the field, but it became apparent that many nuclear proteins do not contain classical mono- or bipartite NLSs and must either use alternate entry mechanisms or piggyback on cargo that does contain a classical NLS. One example is hnRNPA1. This abundant protein shuttles efficiently between the nuclear and cytoplasmic compartments, but the sequence responsible for shuttling, called M9, is glycine and asparagine rich and does not bind to importinα (Table ) (155
). Rather, M9 was shown to be recognized by a novel protein, named transportin (karyopherinβ2), that is related to importinβ (196
). A similar protein, Kap104, was also found in budding yeast (5
) (Table ). Each protein from this family that has been studied to date functions as a carrier in nucleocytoplasmic transport.
A priori, one might have expected that the carriers would be capable of transporting cargo in both directions through the pores, but to date this property has been demonstrated for only two family members, the yeast Kap142/Msn5 and mammalian importin13(157
). All of the other carriers appear to function exclusively either as importins or as exportins. Kap142 can import the yeast trimeric replication protein A (RPA) to the nucleus. As an exportin, it carries various proteins—Pho4, Mig1, Far1, and Ste5—to the cytoplasm (52
). It is unusual in that it exports only phosphorylated cargoes, but the consensus sequence context that allows recognition of specific phosphoserines by this carrier has not yet been defined.
Crm1 (exportin1, or Xpo1 in budding yeast) was the first export carrier to be identified (69
). It recognizes a short motif rich in leucine or related hydrophobic residues, which is found in the protein kinase A inhibitor PKI, in the human immunodeficiency virus (HIV) protein Rev, in RanBP1, and in dozens of other proteins (65
is the prototypical nuclear export signal (NES) sequence (Table ), but other hydrophobic residues can substitute for several of the Leu residues, the number of intervening residues is somewhat variable, and prolines situated between the hydrophobic residues disrupt function (25
). There are also efficient NESs that do not conform even to this rather vague consensus. The NES in the NFAT transcription factor, I
TT, is one example (123
). Additionally, motifs that exactly match the PKI/Rev pattern sometimes have no export function, for instance in Ste5 (unpublished observation), perhaps because they are not exposed on the protein surface. The unambiguous definition of an NES is confounded by the high frequency of hydrophobic residues in protein sequences.
Many other import or export signal sequences must exist, but so far they have not been carefully dissected and are often much larger than the classical NLS and NES motifs. The import signal for uridine-rich small nuclear ribonucleoproteins (U snRNPs) comprises both the m3G cap on the RNA of the U snRNP and sequences within the Sm core protein of the RNP, which are recognized by an adapter protein called snurportin that, like importinα, binds to importinβ (100
). Similarly, the export signal for snurportin, which is recognized by Crm1, is a large domain that encompasses most of the protein rather than a short hydrophobic sequence (185
Most known import signals, except for M9, do have a basic character, however. For example, the NLSs present in ribosomal proteins such as L25 and L23a are highly basic (105
). Some viral cargoes bind directly to importinβ rather than through an importinα adapter, and they also possess highly basic, arginine-rich NLSs (88
). Additionally, certain cargoes with short basic sequences are able to bind to a distinct site on importinα and be transported simultaneously with proteins possessing a monopartite NLS (K. Plafker and I. G. Macara, unpublished data). This common theme may reflect the evolutionary relationship between the importin family and the signals they recognize. On the other hand, it is just as likely to be a quirk that reflects the biased nature of the very small group of NLSs that have been characterized to date.
To add to this complexity, there are transport factors that are unrelated to the importin family and proteins that can translocate through the NPCs in the absence of other soluble factors. In the first category are the yeast protein Mex67 and its mammalian homologue TAP, which most probably bind directly to mRNA sequences (84
); cytoplasmic calreticulin is an export carrier for steroid receptors (96
) (see next section). In the second category there are proteins such as hnRNPK and β-catenin, both of which most probably interact with the NPCs directly (59
Clearly, we have not yet exhausted the repertoire of nuclear import and export signals. Nor have we identified all of the diverse mechanisms by which these signals can be regulated. Numerous proteins possess classical NLSs that are exposed or hidden or show altered import rates when nearby serine or threonine residues are phosphorylated (for a review, see reference 109
). For example, phosphorylation of Pho4 not only permits recognition by Msn5 but also inhibits binding to the importin for this protein, Pse1 (Kap121) (110
). Phosphorylation by the PKB/Akt protein kinase within an NLS in the forkhead transcription factor, AFX, blocks nuclear import and results in a rapid shift of the protein to the cytoplasm (29
). Masking of the NLS in NF-κB by IκB maintains the NF-κB in the cytoplasm until IκB is phosphorylated and degraded. This story is complicated, however, by the discovery that IκB itself shuttles in and out of the nucleus (219
). Another example of masking involves the 14-3-3 family of proteins, which recognize phosphorylated serine residues within the sequence context RSxS
xP (where the asterisk indicates the phosphorylated residue). Cdc25, a protein tyrosine phosphatase that regulates the cell cycle, becomes associated with 14-3-3 when phosphorylated by a checkpoint kinase. Cdc25 possesses both an NES and a dominant NLS, but when bound to 14-3-3 the NLS is masked and Cdc25 accumulates in the cytoplasm (167
). A similar mechanism may be involved in the cytoplasmic retention of forkhead transcription factors.
NESs can also be masked. One example is provided by telomerase reverse transcriptase, to which 14-3-3 can bind in a non-phosphoserine-dependent manner and block an NES (231
). Another case is the transcription factor NFAT, which, in the presence of calcium ions, interacts with calcineurin. NFAT contains both an NES and one or two NLSs (depending on the isoform) and shuttles constitutively, so that masking of the NES by calcineurin permits nuclear accumulation (288
). A twist on this story is that NFAT can also be negatively regulated by protein kinase A, which creates 14-3-3 binding sites on NFAT that may mask the NLS (167
A reverse of this type of situation occurs when NLS or NES function is not masked but requires the formation of a complex. As an illustration, the import of a fission yeast protein called Mei2, which is necessary for meiosis, has to associate with a small RNA (mei-RNA) before it can accumulate in the nucleus (278
). A different mechanism controls the localization of yAP1, a transcription factor that responds to oxidative stress. Cysteine residues within the NES of yAP1 are sensitive to the redox state of the cell, and their oxidation blocks interaction with the exportin, Crm1 (279
Only during the last few years has the extraordinary range of processes that are controlled at the level of nucleocytoplasmic transport been recognized. Everything from apoptosis to circadian rhythms and from signal transduction to the cell cycle is regulated, at least in part, by switching NLSs and NESs on or off. Nuclear transport is the one common link between these diverse processes.