Cell, Vol. 64, 489-497, February8, 1991,Copyright© 1991 by Cell Press

How Proteins Enter the Nucleus

Pamela A. Silver Department of Molecular Biology Princeton University Princeton, New Jersey 08544

The nucleus of eukaryotic cells contains a distinct set of proteins necessary for the management of genes. Following synthesis in the cytoplasm, proteins enter the nucleus through pore complexes, large proteinaceous structures in the nuclear envelope. Certain proteins are transported to the nucleus if they contain an active nuclear localization sequence (NLS). To understand how proteins are localized to the nucleus, we must determine how NLSs are recognized, how proteins containing NLSs are brought to the nuclear pore complex, and how the pore complex mediates the selective entry of NLS-containing proteins. I will review the derivation of our knowledge of the nuclear import apparatus and nuclear protein import as a novel form of regulation. Other recent reviews document NLSs (Garcia-Bustos et al., 1991) and the structure of the nucleus (Newport and Forbes, 1987; Gerace and Burke, 1988). A Selective Transport Model for Nuclear Protein Import The diversity of biochemical reactions in the nucleus necessitates the movement of large numbers of macromolecules. For example, in a growing cell, histones sufficient to double the chromatin content must be imported with each cell division. This means that each pore might transport, on the average, 100-500 histone molecules per minute, as well as all the other necessary nuclear proteins. Protein transport at the pore differentiates the nucleus from other membrane-bound organelles, where the site of entry is not known and where proteins often partially unfold during import. A simple scheme for the localization of proteins to the nucleus is presented in Figure 1. By this model, proteins move to the nucleus and bind at the nuclear pore complex. Cytoplasmic binding proteins may recognize NLSs and deliver proteins to the pore. Following binding at the pore complex, the pore opens and proteins pass through. ATP is needed for movement of proteins into the nucleus. Proteins may be released from or transported in association with NLS-binding proteins. If they are cotransported, once in the nuclear interior, the imported protein would be released and the NLS-binding protein could recycle to the cytoplasm. Various aspects of this model will be considered as the results of current experiments are discussed. Short Stretches of Amino Acids Direct Proteins to the Nucleus Our current view of the nuclear import process derives from the discovery of NLSs, first suggested by De Robertis et al. (1978). Two criteria define NLSs: deletion or mutation causes cytoplasmic accumulation of a normally nu-

Review

clear protein; and when fused to a nonnuclear protein, the NLS directs the protein to the nucleus. The first demonstration that the signal for nuclear import could be restricted to a short, contiguous stretch of amino acids came from studies on nucleoplasmin, the major nuclear protein of the Xenopus oocyte. Dingwall et al. (1982) demonstrated that nucleoplasmin missing its C-terminal tail will not enter the nucleus when introduced into the cytoplasm by microinjection, but remains nuclear when injected directly into the nucleus. However, the C-terminal peptide alone is efficiently transported into the nucleus. This experiment demonstrates that the C-terminal portion of nucleoplasmin is necessary for passage of the complete protein into the nucleus, indicating that a subset of amino acids can act as a nuclear determinant. These results rule out selective nuclear retention as the sole mechanism of nuclear accumulation of nucleoplasmin. Subsequent studies of nuclear uptake of proteins lent overwhelming support to the notion that specific NLSs lie in the transported protein. For instance, a single amino acid change in SV40 T antigen (Lys-128 to Thr or Asn) renders the protein nonnuclear in vivo (Lanford and Butel, 1984; Kalderon et al., 1984). This finding led to the suggestion that the sequence Pro-Lys-Lys-Lys-Arg-Lys-Val could direct a protein to the nucleus. As predicted, this peptide was sufficient to promote nuclear localization of the normally cytoplasmic protein, pyruvate kinase (Kalderon et al., 1984). However, recent results indicate that fully efficient nuclear localization of SV40 T antigen requires more than this minimal sequence. Rihs and Peters (1989) suggest that 15 amino acids immediately preceding the NLS do not form a second NLS but increase the efficiency of the existing NLS. Known phosphorylation sites in this region may affect the function of the T antigen NLS. Robbins et al. (1991) further show that the nucleoplasmin NLS is composed of two interdependent regions of basic amino acids; mutations in either alone have no effect on nuclear localization activity. The nucleoplasmin NLS is nonfunctional only when both domains are mutated. This is similar to what has been shown for the histone H3/H4-binding protein N1 of Xenopus (Kleinschmidt and Seiter, 1988) and polymerase 1 of influenza virus (Nath and Nayak, 1990). The position of the NLS and its context in the transported protein can be important (Roberts et al., 1987; Nelson and Silver, 1989). For example, placement of the SV40 T antigen NLS in the buried hydrophobic domain of pyruvate kinase creates a nonnuclear protein, indicating that the NLS must be exposed on the surface of the protein to interact with components of the import machinery. NLSs can also function outside the linear array of a protein sequence. Bovine serum albumin (BSA) or colloidal gold particles conjugated to synthetic NLS-containing peptides will efficiently migrate into the nucleus, either in vivo following microinjection (Feldherr et al., 1984; Richardson et al., 1988) or in vitro into rat liver nuclei in an extract from Xenopus eggs (Newmeyer and Forbes, 1988).

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PORE COMPLEX

6

ATP

@

I NUCLEOPLASMI

the nucleus via cotransport with another protein (e.g., Dingwall et al., 1982; Zhao and Padmanabhan, 1988). In each case at least one NLS is necessary for import of the protein complex. This supports the notion that some proteins remain in complexes and do not disassemble or unfold as they enter the nucleus. In sum, experiments with genetically engineered fusion proteins, chemically synthesized peptides conjugated to gold particles, and protein-peptide conjugates focus on the properties of the transported protein. They provide compelling evidence that short stretches of amino acids can confer specific nuclear accumulation upon a protein. However, the presence of such sequences does not address the question of whether their binding sites are in the nuclear interior, on the nuclear envelope, in the cytoplasm, or in all compartments. They do, however, imply the existence of a specific apparatus that recognizes NLSs.

Recognition of Proteins for Import

[NUCLEAR ENVELOPE

Figure 1. Model for Entry of Proteins into the Nucleus Following synthesis in the cytoplasm, NLS-containing proteins associate with the nuclear pore complex (1), possibly via NLS-binding proteins at the pore complex (NBP) and/or nucleoporins (NP) or with cytoplasmic NLS-binding proteins (la), and move to the nuclear pore complex (lb). Proteins are translocated through the pore in an ATPrequiring process (2), either after release from (3) or still associated

with NLS-bindingprotein (4). In the latter case, NLS-bindingproteins might be released(5) and recycledto the cytoplasm(6). NLS-binding proteins, nucleoporins,binding of imported proteinsat the pore, and ATP-dependent translocation through the pore have been demonstrated experimentally.The other steps are largely conjecture.

Numerous similarly defined sequences have been identified in nuclear proteins. Comparisons of these sequences has led to the proposal that the SV40 T antigen NLS is the prototypic NLS. However, not all NLSs are of the SV40 T antigen type (e.g., Hall et al., 1984; Silver et al., 1988), although most are rich in basic amino acids. Some proteins contain more than one functionally redundant NLS. For instance, nuclear localization of polyoma large T antigen is eliminated entirely only when both of its NLSs (which are not identical) are mutated (Richardson et al., 1986). Influenza virus NS1, yeast MATe2., and the glucocorticoid receptor are proteins with two NLSs (reviewed in Garcia-Bustos et al., 1990). Multiple NLSs may increase the efficiency with which proteins interact with the NLS-binding sites at the earliest step in transport into the nucleus. Dworetzky et al. (1988) found that the rate of uptake of microinjected protein-coated gold particles increased as a function of the amount of SV40 T antigen NLSs per gold particle. Some proteins do not possess their own NLS and enter

Experimental evidence for the existence of a nuclear transport receptor was provided by Goldfarb et al. (1986), who cross-linked BSA to the SV40 T antigen NLS peptide and found that its rate of uptake was saturable. Further support for receptor-mediated uptake comes from findings indicating that transport is at least a two-step process (Newmeyer and Forbes, 1988; Richardson et al., 1988). Import into nuclei, assayed in vitro, is dependent on temperature, ATP, and a functional NLS. In the absence of ATP, proteins bind at the nuclear pore complex but are not imported. In vivo studies indicate that binding may occur first in the cytoplasm as well as at the nuclear periphery, and could be disassociated from translocation (Richardson et al., 1988; Breeuwer and Goldfarb, 1990). Thus, proteins that recognize NLSs may first bind in the cytoplasm and deliver proteins to the pore complex. There also may be multiple sets of NLS-binding proteins, some in the cytoplasm and some at the nuclear surface (Adam et al., 1989; Yamasaki et al., 1989; see below). The diversity of peptides that can direct proteins to the nucleus suggests that multiple proteins might recognize varying classes of NLSs. Alternatively, one protein might interact with all NLSs. Both alternatives are still formally possible. Proteins have been identified from a number of sources that each interact with several different NLS peptides. A summary of the properties of the known NLSbinding proteins follows. Table 1 contains a current list, but further studies are needed to determine if any (or all) of these proteins are related. Peptides corresponding to several NLSs bind to polypeptides of approximately 60 and 70 kd in rat liver cells (Adam et al., 1989; Benditt et al., 1989; Yamasaki et al., 1989; Imamoto-Sonobe et al., 1990) and 66 kd in HeLa cells (Li and Thomas, 1989). These proteins were identified with antibodies and by chemical cross-linking to synthetic NLS peptides. The NLS binding is specific, since the same proteins do not recognize an import-defective mutant peptide. These NLS-binding proteins have been found in cytosol and nuclei and at the nuclear envelope. Two additional rat liver proteins of 140 kd (nuclear) and 100 kd (cytoplasmic) have also been identified (Yamasaki et

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Table 1. Componentsof the Nuclear Import Apparatus Protein

Organism

Subunit Size ( k d )

Location

Nucleporins: p62 NUP1 NSP1 NLS-binding proteins

Rat, mouse, xenopus Yeast Yeast

62 130 110

Nuclear pore complex Nuclear pore complex Nuclear pore complex, nuclear envelope

Rat, yeast Rat, yeast Rat, yeast Human, rat, yeast Rat, yeast

138-140 97-98 76 66-70 56-59

Nucleus Cytoplasm, nucleus Nuclear envelope Cytoplasm, nuclear envelope, pore complex Cytoplasm, nuclear envelope, nucleus

Other: Gp190 (210)

Rat, dog, chicken, Drosophila

190 (- sugar) 210 (+ sugar) ? ? 73 188

Nuclear pore complex Cytoplasm Cytoplasm Endoplasmic reticulum, nuclear envelope Nuclear envelope

NIF-1 NIF-2 NPLl/SEC63 Myosin-like ATPase

Xenopus Xenopus Yeast Drosophila,rat

The identificationand possible function of the various components are discussed in the text.

al., 1989). All four proteins bind most NLSs tested, albeit with different relative affinities. In the yeast Saccharomyces cerevisiae, there are two major NLS-binding polypeptides of about 70 kd and 59 kd (and minor proteins of 140 kd and 97 kd) associated with the nucleus. NLS-binding proteins were identified in yeast by binding of NLS peptides to proteins immobilized on nitrocellulose (Silver et al., 1989; Lee and Melese, 1989). These proteins bind not only the SV40 T antigen NLS, which has been shown to function in yeast as it does in animal cells (Nelson and Silver, 1989), but also to NLSs from histone H2B, GAL4, and nucleoplasmin. These proteins can distinguish between normal and mutated SV40 T antigen NLS peptides, suggesting NLS binding specificity. Thus, yeast and mammalian cells share 66-70 kd and 59-60 kd nucleus-associated proteins that recognize NLSs. How do certain proteins recognize NLSs? Mutations in NLSs demonstrate that a high content of basic amino acids is not sufficient for nuclear import. Replacement of Lys-128 of the SV40 NLS with other negatively charged amino acid derivatives does not restore full activity (Lanford et al., 1988), and a peptide corresponding to the SV40 NLS with the amino acids in the reverse order does not bind to the receptors from rat liver (Adam et al., 1989). However, in a general sense, NLSs are similar to other signal sequences: in no case is there a good primary sequence consensus. Those for secretion are generally hydrophobic, those for mitochondrial import are a combination of basic and hydrophobic residues, and NLSs are generally basic. Similarly, transcriptionally activating acid blobs are all acidic but have little primary sequence homology. All of these cases may be examples of the recurring theme of semispeciflc interactions; many ligands are allowed, but other potential ligands are excluded. How do NLS-binding proteins function in nuclear localization? One possibility is that they are part of the nuclear pore complex. Binding of imported proteins at the pore

complex prior to import would seem to support this view (Newmeyer and Forbes, 1988). On the other hand, NLSbinding proteins could interact with proteins in the cytoplasm and deliver them to the pores. When nuclear import is inhibited by depletion of ATP, nuclear proteins accumulate not only on the nuclear surface, but also in the cytoplasm (Richardson et al., 1988; Breeuwer and Goldfarb, 1990). The transported protein might remain attached to the NLS-binding protein as it is translocated through the pore. Release could occur at the pore complex or in the nuclear interior. In the latter case, the NLSbinding protein would recycle to the cytoplasm (Borer et al., 1989). Alternatively, there could be two sets of NLSbinding proteins, one for delivery to the nucleus and the other for retention once the proteins have passed through the pore. All of the above are possible because NLSbinding proteins have been found in all relevant compartments, i.e., cytoplasm, nuclear envelope, and nucleoplasm. Nuclear protein import in permeabilized mammalian cells requires factors present in a cytoplasmic extract (Adam et al., 1990). One N-ethylmaleimide-sensitive cytosolic factor, termed NIF-1, is necessary for correct import of proteins into rat liver nuclei placed in an extract from Xenopus eggs and is necessary for binding of proteins to the nuclear pore complex (Newmeyer and Forbes, 1990). A second N-ethylmaleimide-sensitive cytosolic factor, NIF-2, stimulates the effect of NIF-I. Examination of import into isolated nuclei from yeast does not reveal a requirement for a similarly defined cytoplasmic factor (Kalinich and Douglas, 1989). NIF-1 and NIF-2 may be part of a fiber-based network along which macromolecules are transported to and/or through the pores (Newmeyer and Forbes, 1990). Proteins conjugated to gold particles line up at the pore prior to translocation, suggesting some association with fibers (Feldherr et al., 1984; Richardson et al., 1988). However, the NLS-binding proteins are only loosely associated with the nucleus or distributed between the nucleus and the

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cytoplasm and are unlikely to be part of such a fibrous system (Silver et al., 1989; Adam et al., 1989; Yamasaki et al., 1989). The Nuclear Pore Complex Is the Site of Entry and Exit of Macromoleculea The pore complex, the major hallmark of all nuclear envelopes from fungi to man, is the site of protein entry into and RNA exit from the nucleus. It is formed by a large proteinaceous complex of about 108 daltons that perforates both the inner and outer nuclear membranes (Reichelt et al., 1990, and references therein). When visualized by electron microscopy, the nuclear pore complex is 1200-1500 .~ in diameter and eight-fold rotationally symmetric. The structural features of the pore complex are two "rings;' each of eight subunits, on the inner and outer surfaces of the nuclear envelope, and "spokes" projecting inward toward a 90 ,~, aqueous channel, which is often occupied by a particle or central granule (reviewed in Gerace and Burke, 1988). Much of our knowledge about the transport properties of the nuclear pore is from microinjection of molecules into the cytoplasm and examination of their subsequent distribution (reviewed by Peters, 1986). Nonnuclear proteins smaller than approximately 40-60 kd often equilibrate between the nucleus and cytoplasm, while larger proteins are excluded from the nucleus, confirming the presence of an aqueous channel. In contrast, following introduction into the cytoplasm, nuclear proteins, regardless of size, enter and concentrate in the nucleus. These observations indicate that nuclear association of the injected proteins is not coupled to protein synthesis or cotranslational modifications such as proteolytic processing (Dabauvalle and Franke, 1982). The mature protein contains sufficient information for its nuclear import. Feldherr et al. (1984) examined the behavior of gold particles coated with the nuclear protein nucleoplasmin or with peptides corresponding to NLSs from SV40 T antigen upon injection into the cytoplasm of the Xenopus oocyte. The particles enter the nucleus only at the pores. When coated with nucleoplasmin lacking its NLS or with mutated forms of the NLS peptide, gold particles remain in the cytoplasm. These experiments indicate that import occurs at the pore and that the pore complex is sufficiently flexible to accommodate the passage of a particle as large as 250 ,~, (the size of some of the largest nucleoplasmincoated gold particles) in response to an NLS. Prior to entering the nucleus, the protein-coated gold particles align at the cytoplasmic surface of the nuclear envelope and often appear to associate with fibrils emanating from the pore complex (Feldherr et al., 1984; Richardson et al., 1988; Newmeyer and Forbes, 1988). One of the earliest steps in import may be the association of proteins with a network of fibers that span the pore (Georgatos and Blobel, 1987). More detailed analysis of the transport of nucleoplasmin-gold conjugates has revealed binding at multiple sites around the periphery of the nuclear pore complex as well as association with the central plug, which some authors refer to as the "transported' (Akey and Goldfarb, 1989). Using cryoelectron

microscopy, Akey (1990) has described nuclear pore complexes in four conformations that may correspond to different stages in protein transport. A model with two opposing iris-like structures in the plane of the nuclear envelope has been proposed to explain opening of the pore. However, these results present only static images and may suffer from redistribution of proteins during sample preparation. The nuclear pore must accommodate not only the entry of large proteins but also the egress of particles as large as ribosomes, which are assembled at the nucleolus. RNA-coated gold particles of up to 230 ~, in diameter, when injected into the nuclear interior, exit the nucleus only at the pores (Dworetzky and Feldherr, 1988). Export is temperature dependent and saturable. However, it is unclear whether there is sequence specificity for this process, since gold particles coated not only with tRNA, 5S RNA, or poly(A), but also those coated with nonphysiological polynucleotides such as poly(dA) or poly(I), are all exported. Ribosomal RNAs probably exit the nucleus in association with certain proteins, which may account for their specific translocation. 5S rRNA is transported to the cytoplasm only if it can bind to TFIIIA or ribosomal protein L5. Mutant rRNAs that do not bind to these proteins remain in the nucleus (Guddat et al., 1990). With regard to protein entry, the same pore can accommodate both RNA exit and protein import (Dworetzky and Feldherr, 1988). Perhaps the two processes will be found to use some of the same components. Proteins of the Nuclear Pore Complex The nuclear pore complex could be considered an organelle composed of a unique set of proteins necessary for transporting macromolecules across the nuclear envelope. Association of the nuclear pore complex with both the nuclear membrane and the underlying lamina has made it difficult to isolate in pure form. Hence, only a subset of nuclear pore complex-associated proteins have so far been identified (Gerace and Burke, 1988, and references therein). One set of pore complex proteins (termed nucleoporins) are O-glycosylated with N-acetylgluocosamine (GIcNAc) and thus interact with the lectin wheat germ agglutinin (WGA [see Hart et al., 1989, for review]). Unlike other sugar modifications, O-linked GIcNAc addition occurs in the cytoplasm shortly after or during protein synthesis and prior to assembly into the pore complex (Davis and Blobel, 1987). Some nucleoporins are located on the nucleoplasmic surface, while others are on the cytoplasmic surface of the pore complex. Three lines of evidence indicate that the some nucleoporins play a specific role in protein translocation. First, coincident labeling of nuclear pore complexes with WGAgold and nucleoplasmin-gold particles suggests that nucleoporins interact directly with proteins prior to their passage through the pore (Akey and Goldfarb, 1989). Second, antibodies that recognize a subset of nucleoporins block nucleoplasmin import and RNA export (Featherstone et al., 1988). And third, WGA blocks uptake of pro-

Review:NuclearImport 493

teins into the nucleus (Yoneda et al., 1987; Dabauvalle et al., 1988). The effect of WGA is not due to occlusion of the channel, because diffusion of dextrans through the pore is not blocked (Finlay et al., 1987). Instead, pore complexes assembled in vitro without WGA-binding proteins are morphologically intact but are unable to import large proteins correctly (Finlay and Forbes, 1990). These reconstituted pores fail to bind NLS-bearing proteins. Addition of WGA-binding pore complex proteins restores binding and import. However, the nucleoporins with the O-linked GIcNAc modification probably do not include NLS-binding proteins, because those from rat do not interact with WGA (Yamasaki et al., 1989). Instead, nucleoporins may provide the docking site for NLS-binding protein complexes. Genes encoding three nucleoporins, p62 of rat and NSP1 and NUP1 of yeast, have been cloned and sequenced. Their sequence features suggest that these are structural proteins characterized by simple repeated motifs. All of these proteins were initially identified with antibodies; this approach may selectively identify more abundant proteins as well as proteins with repeated antigenic sequences. p62 has three apparent structural domains: an N-terminus with a repeated tetrapeptide, a central region rich in serine and threonine, and a C-terminus composed of hydrophobic heptad repeats characteristic of coiled coils of ~ helices in several filament-forming proteins. One O-linked GIcNAc addition site has been identified, and p62 probably contains eight to ten such sugar residues (Starr et al., 1990). Proteins of the yeast S. cerevisiae that are recognized by antibodies against the mammalian p62 nucleoporin (Aris and Blobel, 1989) are of approximately 100 kd and reside at the yeast nuclear pore complex. The NUP1 gene, encoding one of these nucleoporins, predicts a protein sequence containing a series of repeats similar to those found in the center of another previously identified yeast nucleoporin, NSP1 (Davis and Fink, 1990; Nehrbass et al., 1990). These repeated sequences may be a common feature of many nucleoporins. In NSP1, copies of the repeated motif KPAFSFGAK are separated by a 10 amino acid spacer that is somewhat less conserved and consists almost entirely of charged and hydrophilic amino acids. In NUP1, this sequence is less well conserved, and the spacer regions are more variable in length and composition. (There is no primary sequence homology between NUP1 and the rat p62, suggesting that the anti-p62 monoclonal antibodies recognize some secondary structure in NUPI.) The C-terminus of NSP1, like the mammalian p62 C-terminus, contains typical amino acid heptad repeats and is the only part of the protein that is essential for cell viability (Nehrbass et al., 1990). It may be that the repeated units in the N-terminus and center of this protein can be replaced by other stretches of amino acids with similar motifs. At least one mammalian pore complex-associated integral membrane glycoprotein has been identified and cloned (Gerace and Burke, 1988; Wozniak et al., 1989; Greber et al., 1990). The gene sequence predicts a 204 kd polypeptide with a cleavable signal sequence for tar-

geting to the endoplasmic reticulum membrane and two potential membrane-spanning regions. The native protein contains N-linked high mannose oligosaccharides, bringing its total mass to ,'~210 kd. Gp210 may serve to anchor pore complex proteins at the membrane and/or to catalyze fusion of nuclear envelope membranes to create sites for nucleation of new pores. However, there is no direct evidence for the role of Gp210 in nuclear import. In summary, proteins can enter the nucleus via the nuclear pore. The pore complex is a dynamic structure that probably opens and closes in a regulated manner and is composed of many proteins (only a few of which have been characterized). The problem of enumerating the components and understanding the structure of the nuclear pore complex may be similar or greater in scale to understanding the composition of the ribosome. The approach of isolating proteins based on location in the pore complex could eventually reveal all the components, but functional assays such as those used to identify NLSbindng proteins and reconstitute functional pore complexes will also be valuable. Nuclear Protein Import Is an Active Process

The requirement for ATP has been used to define two steps for nuclear protein import. Following binding at the pore complex or the outer nuclear membrane, ATP is required for subsequent translocation into the nucleus. In vitro, depletion of ATP inhibits nuclear import but not binding at the pores (Newmeyer and Forbes, 1988). In vivo, proteins also accumulate on the nuclear envelope (Richardson et al., 1988) and in the cytoplasm (Breeuwer and Goldfarb, 1990) after ATP depletion. Addition of ATP rastores pore-dependent import. Little more is known about how ATP catalyzes import. A myosin heavy chain-like ATPase is associated with Drosophila embryo and rat liver nuclear envelopes, indirectly suggesting that hydrolysis of ATP is important for pore function (Berrios and Fisher, 1986). For instance, ATP hydrolysis might be required for release of NLS-containing proteins from their binding sites once they arrive at the pore. Release would then allow the proteins to move through the pore. Early models for nuclear localization suggested that proteins could enter the nucleus by diffusion and then be retained within the nucleus through interaction with some component within the nucleus, such as DNA, or after assembly into larger structures such as the nuclear lamina. This model, however, cannot explain entry of molecules larger than the diffusion limit size of the pore. Instead, more recent models proposing active transport of nuclear proteins to and through the pore complex unify the existing data. However, the possibility still exists that proteins smaller than the estimated diameter of the aqueous channel may simply enter the nucleus by diffusion. Experiments with variously sized dextrans and proteins microinjected into the cytoplasm have shown that molecules smaller than ,~60 kd can enter the nucleus by diffusion (reviewed in Peters, 1986). However, certain small proteins such as histone H2B (,~15 kd) contain a NLS (Moreland et al., 1987), and histone H1 (,~,21 kd) enters the nucleus in a temperature- and energy-dependent manner, sug-

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gesting active import for these small proteins (Breeuwer and Goldfarb, 1990). However, histones may exist in complexes with other NLS-bearing proteins such as nucleoplasmin and N1 (Kleinschmidt et al., 1985; Dilworth et al., 1987) and enter the nucleus as large protein complexes. The cytoplasm contains many small proteins that are excluded from the nucleus. Perhaps these proteins contain "signals" to keep them in the cytoplasm; for example, they may simply form loose complexes with other cytoplasmic proteins (Dabauvalle and Franke, 1984). Microinjection experiments to assess pore permeability are often done with normally extracellular proteins or nonphysiological molecules such as dextrans. For instance, cytochrome c (,~15 kd) apparently does diffuse into the nucleus following microinjection into the cytoplasm (Breeuwer and Goldfarb, 1990). However, fusion of an NLS to cytochrome c renders its import into the nucleus both temperature and energy dependent. Similarly, fusion of the SV40 NLS to cytochrome cl targets the protein to the nucleus in yeast (Sadler et al., 1989). Proteins like cytochrome Cl that are destined for organelles other than the nucleus contain targeting signals specific to the organelle. Thus, the diffusion of exogenous proteins such as cytochrome c into the nucleus may be irrelevant because these proteins may normally be moving in a vectorial manner through the cytoplasm. The question remains open as to whether all protein traffic into the nucleus is actively mediated by NLSs or if diffusion accounts for the natural distribution of any protein between the nucleus and the cytoplasm. Taken together, the results of many types of experiments support the concepts of selective recognition and active transport of proteins into the nucleus (Figure 1). But we are left with many uncertainties: Why are there multiple NLS-binding proteins that recognize the same NLSs? If proteins are cotransported with an NLS-binding protein, how are they released once inside the nucleus? Do some pore complex proteins act as receptors for NLS-binding proteins? The answers will either refine the current model or suggest new ones. In summary, relatively few factors have been identified as important for passage of proteins into the nucleus (summarized in Table 1). The precise role of most of these factors has not been defined. Given the complicated nature of the process, this short list will undoubtedly grow rapidly. New approaches will further delineate components of the nuclear import apparatus. For example, yeast mutants have been isolated that have a defect in nuclear protein localization (npl mutants [Sadler et al., 1989]). Identification of nuclear localization mutants relies on the missorting of a normally nuclear-targeted protein to another cell compartment, in this case the mitochondria. One of these mutants, npll, corresponds to a gene, SEC63, that is also necessary for correct assembly of proteins into the endoplasmic reticulum. SEC63 encodes a membrane protein and may be important for the assembly or correct functioning of the pore complex. Mutations in at least five other NPL genes have been identified that affect nuclear protein localization in yeast.

Regulation of Nuclear Protein Import Is Achieved by Masking of NLSs The activity of some proteins is regulated by their transport from the cytoplasm to the nucleus at a certain stage of the cell cycle or in response to developmental cues or growth factors. How nuclear import of these proteins is controlled is not understood in detail. I summarize what is known about a few cases of regulated nuclear protein import. The glucocorticoid receptor is a zinc finger-type DNAbinding protein that regulates transcription of genes in response to certain steroid hormones. In the absence of glucocorticoid, the glucocorticoid receptor is cytoplasmic. In the presence of hormone, the receptor rapidly translocates into the nucleus where it binds to DNA and performs its regulatory role. Translocation into the nucleus depends on interaction of the receptor with hormone. The NLSs within the receptor (Picard and Yamamoto, 1987) are nonfunctional in the absence of hormone because they are obscured by the heat shock protein, hsp90, bound to glucocorticoid receptor in the cytoplasm (Sanchez et al., 1985). By one model, hormone binding to the receptor dissociates it from hsp90 and the NLSs become exposed, which then results in glucocorticoid receptor translocation into the nucleus. However, hormone binding is required not only for nuclear localization but also for glucocorticoid receptor activity (Picard et al., 1988). Thus, receptor action is not regulated solely at the level of its location. In the Drosophila embryo, the maternally supplied dorsal protein controls the asymmetric expression of zygotic genes. In cleavage stage embryos, the dorsal protein is distributed throughout the cytoplasm. When the nuclei migrate to the embryo surface, dorsal protein on the ventral side enters nuclei, forming a ventral-to-dorsal nuclear concentration gradient (Rushlow et al., 1989; Steward, 1989; Roth et al., 1989). Analysis of the distribution of the protein in mutant embryos suggests that the dorsal protein is inactive in the cytoplasm and that the activity of dorsal protein is regulated at the level of its nuclear localization. NF-KB is a ubiquitous mammalian transcription factor whose activity is regulated at the level of its intracellular location (Baeuerle and Baltimore, 1988). In stimulated B lymphocytes, NF-KB is nuclear, binds DNA, and regulates the transcription of K immunoglobulin light chain genes. However, in pre-B cells, where K light chains are not expressed, NF-KB is cytoplasmic. The cytopia,smic form of NF-~:B is associated with another protein, IKB, and this complex is unable to bind DNA. Phosphorylation disrupts the IKB-NF-KB complex and NF-KB enters the nucleus (Ghosh and Baltimore, 1990). As for glucocorticoid receptor, association with cytoplasmic IKB may block the activity of the NF-KB NLS. NF-KB, dorsal, and the rel oncogene have a high degree of sequence similarity extending over 300 amino acids in the N-terminal half of the proteins (Gilmore, 1990, and references therein). All three proteins have an SV40 T antigen-like NLS toward the end of the region of homology. In addition, all three proteins have a conserved serine approximately 20 amino acids before the nuclear Iocaliza-

Review:NuclearImport 495

tion sequence. One intriguing possibility is that phosphorylation at this serine mediates nuclear localization either by disrupting the interaction with cytoplasmic factors or, conversely, by effecting the NLS activity by altering the local charge density or the conformation of the protein. Mutation in the cactus gene of Drosophila results in ventralization of all the cells in the embryo. In these mutants, dorsal protein is now nuclear throughout the embryo (Steward, 1989; Roth et al., 1989). cactus protein may work like IKB and interact with dorsal protein in the cytoplasm to prevent its migration into the nucleus. An activity that disrupts this interaction locally would allow dorsal to enter the nucleus mediated by its NLS. In summary, studies of dorsal, NF-KB, the glucocorticoid receptor, and the rel oncoprotein suggest that nuclear localization can be regulated by masking of the NLS. It is not clear how this comes about. One possibility suggested by analysis of the glucocorticoid receptor and NFKB is that binding to a cytoplasmic factor anchors the protein in the cytoplasm and masks the NLS in these proteins so that they are unable to interact with NLS-binding proteins. In Drosophila, candidate genes that may be involved in reversing the inhibition of dorsal nuclear import by cactus have been identified (reviewed in Govind and Steward, 1991).

Regulation of Nuclear Import during the Cell Cycle At different stages of the cell cycle, the nucleus must accommodate varying amounts of exchange of macromolecules between the cytoplasm and the nuclear interior. For instance, at the onset of S phase, the amount of histone in the nucleus doubles. During mitosis, the nuclear envelope of many cells breaks down into vesicles. Many nuclear proteins are dispersed throughout the cell and are rapidly imported into the nucleus at the end of mitosis. Taken together, these observations suggest a high degree of variation in the permeability of the nuclear envelope during the cell cycle. This may occur by varying the number or the activity of pore complexes. Feldherr and Akin (1990) examined the permeability of the nuclear envelope in dividing and confluent cell cultures. Nuclei from dividing 3T3-L1 cells took up nucleoplasmin-coated gold particles at a rate 7 times that of growth-arrested cells. Growth-dependent differences in nuclear permeability could be explained by changes in pore composition and/or structure or overall changes in intracellular energy levels during different growth stages. Blow and Laskey (1988) have proposed that proper replication of DNA during the cell cycle depends on an essential replication factor, termed licensing factor, that is normally unable to cross the nuclear envelope. Instead, they propose that licensing factor enters the nucleus during mitotic breakdown of the nuclear envelope. In this way, licensing factor binds to DNA at certain sites in a temporal manner. By one hypothesis, after DNA replication, licensing factor may be inactivated and DNA is unable to replicate again because active licensing factor is excluded from the nucleus until the next mitotic division. In the yeast S. cerevisiae, where the nuclear envelope

remains visibly intact throughout the cell cycle, the transcription factor SWI5 is localized to the nucleus in a cell cycle-dependent manner (Nasmyth et al., 1990). SWI5 synthesis begins in S phase and lasts through the end of mitosis. The newly made protein remains in the cytoplasm. As cells enter G1, SWI5 rapidly moves into the nucleus to activate transcription of the site-specific HO endonuclease. The data indicate that nuclear entry alone is sufficient to explain the cell cycle regulation of HO expression by SWI5. No cytoplasmic anchoring protein has been implicated in keeping SWl5 out of the nucleus. Instead, nuclear entry may be mediated by a cell cycle-specific modification of SWI5.

Why Do Cells Have a Nuclear Envelope? The role of nuclear permeability in regulation may be relevant to all eukaryotic cells and allows speculation on the evolutionary origin of the nuclear envelope. The nucleus may not have evolved to concentrate proteins at DNA. In Escherichia coli, for example, most of the "free" lac repressor is associated nonspecifically with DNA (Kao-Huang et al., 1977). There would be no obvious selective advantage to forming a barrier such as the nuclear envelope and then requiring concentration of proteins that originally were present at their proper location in sufficient quantities. Instead, we can imagine that the nucleus evolved to selectively keep certain factors out, as indicated by Blow and Laskey (1988) for replication factors. This may have allowed the cell to regulate its cell cycle and level of ploidy in a manner not available to prokaryotes. By importing proteins only at certain times, rapid and discrete changes in intranuclear protein concentrations can be achieved. The nuclear pores themselves may serve as regulators of gene activity by selectively interacting with certain transcribed genes. By the gene-gating hypothesis (Blobel, 1985), transcripts would then be constrained to leave the nucleus only at certain sites because of the nonrandom distribution of pores, thus creating intracellular gradients of RNAs. Nuclear pores may then have arisen as a consequence of gene attachment sites on the prokaryote plasma membrane. Membrane invaginations at the sites of gene attachment may have led to formation of the nuclear envelope.

Summary Nuclear protein import is a selective process. Proteins destined for the nucleus contain NLSs. These short stretches of amino acids interact with proteins located in the cytoplasm, on the nuclear envelope, and/or at the nuclear pore complex. Following binding at the pore complex, proteins are translocated through the pore into the nucleus in a manner requiring ATR The biochemical dissection of the nuclear pore complex has begun. Alteration of protein import into the nucleus is emerging as a new and complex form of regulation. However, we are left with the following problems: How do proteins move through the cytoplasm to reach the nuclear pore? How does the nuclear pore complex open and close in a selective manner? How is ATP utilized during import? And finally, how is bi-

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d i r e c t i o n a l traffic of both p r o t e i n s a n d R N A t h r o u g h t h e pore regulated?

Acknowledgments I especially thank Ruth Steward and Jeff Way for their extensive editorial suggestions; Jim Broach, Douglass Forbes, Werner W. Franke, David Goldfarb, Jim Shepard, Tom Silhavy, Klaus Pfanner, Robin Wharton, Bill Wickner, Martin Wiedmann, Brigitte Wiedmann, and the members of my laboratory for helpful comments; Evelyn Steckman for help in preparing the manuscript; and the support of an Established Investigator Award from the American Heart Association.

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