Plant Physiol. (1999) 119: 1157-1164

UPDATE ON CELL BIOLOGY

Protein Targeting to the Nuclear Pore. What Can We Learn from Plants?¹

Harley M.S. Smith and Natasha V. Raikhel^*

Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312

	INTRODUCTION

Top Introduction References

Characteristic of eukaryotic cells are the numerous types of membrane-bound organelles or compartments found in the cytoplasm, with each type carrying out an essential function for the cell. The spatial separation of proteins and biochemical pathways typical of the various types of organelles requires selective targeting apparatuses. Because each type of organelle contains its own targeting apparatus, proteins destined for a particular organelle must contain the proper targeting signal(s) for entry. These signal-dependent targeting pathways ensure that proteins are targeted to the proper organelle. Understanding how proteins are targeted to the different types of organelles is an important goal in the field of cell biology.

The nucleus is a double-membrane-bound organelle that separates DNA replication and transcription from protein synthesis. Communication between the nucleus and the cytoplasm is a selective process that occurs through large proteinaceous structures called NPCs, which are embedded in the nuclear envelope. Nucleocytoplasmic communication is bidirectional, and it is essential for the cell to know, for example, when to divide and how to respond to various environmental signals. Proteins involved in cell-cycle regulation and transcription factors linked to various signal transduction pathways are examples of proteins that are targeted to the nucleus through NPCs. In addition, the export ribonucleoproteins such as mRNA, tRNA, uridine-rich small nuclear RNAs, and rRNA protein complexes are examples of molecules that exit the nucleus through the NPCs. Therefore, understanding the mechanisms that target proteins and ribonucleoproteins into and out of the nucleus is essential to elucidating communication between the nucleus and cytoplasm.

Several nuclear import and export targeting signals have been identified and characterized in vertebrates and yeast (for reviews, see Görlich, 1997; Nigg, 1997). Each type of signal is linked to an import and/or export pathway. Signal-mediated import and export are facilitated by a family of carrier proteins called the importin -like proteins, in conjunction with a small GTPase, Ran/TC4 (for reviews, see Görlich, 1997; Nigg, 1997). The importin -like proteins function as the nuclear signal receptors for specific import and export pathways; RanGTP regulates the binding of these receptors to the cargo bearing the nuclear- targeting signals.

The classical NLS is a well-characterized targeting signal found in all eukaryotes, including higher plants (for review, see Hicks and Raikhel, 1995b). Transcription factors and cell-cycle regulators are examples of proteins that contain NLSs. Although there is no consensus sequence for NLSs, they have some common features. Typically, NLSs are rich in basic amino acids and are not cleaved after import. In addition, NLSs are position independent; some proteins can contain multiple NLSs. The NLS import pathway is unique in that a heterodimer of importins  and  is required. In this pathway, the importin -subunit functions as the receptor modulator during NLS protein import. These import receptors appear to be conserved in all eukaryotes, including higher plants. In plants recent studies have highlighted a number of unusual features, and as our understanding of import in plants increases, we have gained new insights, such as a model for the targeting of proteins from the cytoplasm to the NPC. These advances will contribute to further expansion of our knowledge of nuclear import in eukaryotes.

	IN VITRO IMPORT SYSTEMS IN VERTEBRATES AND YEAST

To characterize NLS protein import and to identify factors involved in this process, in vitro import systems were developed that use purified nuclei (for review, see Hicks and Raikhel, 1995b) and permeabilized vertebrate or yeast cells (Görlich, 1997; Nigg, 1997). Characterization of the nuclear import process using both systems yielded similar results; however, the permeabilized cell system is favored because the integrity of the nuclear envelope and other structures is preserved in the cell (Görlich, 1997; Nigg, 1997). In the permeabilized cell system, only the plasma membrane is permeable to macromolecules; therefore, under these conditions the soluble contents of the cell become depleted. Import is studied by adding fluorescently labeled NLS substrates to the permeabilized cells, and nuclear import is detected by fluorescence microscopy.

Characterization of the nuclear import process in vertebrates and yeast demonstrates that import can be divided into two distinct stepsdocking and translocation. Docking is an energy-independent step that occurs at the cytoplasmic face of the NPC, whereas translocation through the NPC is energy and temperature dependent. In addition, import is dependent on the readdition of a crude cytosolic fraction in vertebrates and yeast. Biochemical fractionation of the cytosol led to the identification and characterization of proteins involved in this process (Görlich, 1997; Nigg, 1997).

	THE NLS PROTEIN IMPORT PATHWAY IN VERTEBRATES AND YEAST

Protein import occurs when a heterodimer of importin  and  binds NLS-containing proteins in the cytoplasm via the NLS-binding region of importin , forming a trimeric complex (Fig. 1; for reviews, see Görlich, 1997; Nigg, 1997). Docking of the trimeric complex to the cytoplasmic face of the NPC is mediated by the importin -subunit. In yeast and vertebrates, importin  requires importin  for high-affinity interaction with NLSs. Also, importin  can function alone as an import receptor for a subset of NLS proteins.

View larger version (34K): [in this window] [in a new window]   Figure 1. A schematic representation of NLS protein import. The NLS docking step of import is mediated by the importin / heterodimer. The -subunit binds to NLSs and the -subunit mediates NPC docking. Translocation requires RanGDP and GTP. After translocation the import complex docks at the nucleoplasmic side to the NPC. Inside the nucleus, RanGTP binds to importin  and terminates import by releasing the /NLS-containing protein into the nucleoplasm. After importin  dissociates from the NLS-containing protein, it is exported by the Cas/RanGTP complex. Importin  is probably exported with RanGTP. Dissociation of these complexes occurs by the RanGTP-activating proteins RanGap1 and RanBP1, which are found only in the cytoplasm. This allows the formation of the importin / complex, and the cycle of NLS protein import can continue.

Translocation of the trimeric complex through the NPC requires free GTP and a small GTPase, Ran, in the GDP-bound form (Fig. 1). Active in this process are RanGAP1 and RanBP1, which are exclusively localized in the cytoplasm. Recently, it was shown that only the GDP-bound form of Ran can bind to the NPC, but the energy necessary for translocation requires GTP hydrolysis by Ran (Görlich, 1997). These studies suggest that RanGDP must be targeted to the NPC and converted to RanGTP by a nucleotide exchange factor during the import process.

After translocation, the trimeric complex docks at the nuclear basket of the NPC, where the termination step occurs. Inside the nucleus, Ran exists in the GTP-bound form through the action of the guanine nucleotide exchange factor RCC1, which is exclusively located in the nucleoplasm. In vitro binding studies indicate that the binding of RanGTP to importin  terminates import by releasing the importin /NLS-protein complex into the nucleoplasm. Subsequently, importin  dissociates by an unknown mechanism from the NLS-containing protein, and the importin - and -subunits are exported to the cytoplasm, where they can participate in another cycle of import.

Importin  is probably exported to the cytoplasm in a complex with RanGTP. This complex is dissociated in the cytoplasm through the action of importin  and a set of RanGTP-activation proteins, RanGap1 and RanBP1, which are found exclusively in the cytosol. Export of importin  is facilitated by a heterodimer consisting of importin , called Cas, and RanGTP. Cas binds to importin in the presence of RanGTP, creating a trimeric complex that is exported to the cytoplasm. The RanGTP-activating proteins located in the cytoplasm bring about complex dissociation. Cas has a low binding affinity for importin  in the absence of RanGTP, which allows the formation of the importin / heterodimer in the cytoplasm. Thus, the NLS protein import cycle can continue.

	CHARACTERISTICS OF NUCLEAR IMPORT SYSTEMS IN PLANTS

In vitro import systems using permeabilized tobacco protoplasts have been established and used to characterize the NLS protein import pathway in plants. An indirect nuclear import assay was developed using Triton X-100-permeabilized evacuolated parsley protoplasts. Permeabilization allows most cytosolic proteins to leak out of the cell, and it allows large molecules, such as antibodies, to enter the cell. In this assay, antibodies to proteins that are supposed to go to the nucleus are added to permeabilized protoplasts, and import of the antigen-antibody complex is measured by protease protection assays of the intranuclear antibody by immunoblot analysis (Harter et al., 1994).

Recently, an in vitro import system that directly measures NLS protein import was developed and characterized using evacuolated permeabilized protoplasts derived from tobacco suspension cultures (Hicks et al., 1996; Merkle et al., 1996). Typically, vacuoles are rather fragile organelles containing high levels of hydrolytic activity and account for 80% of the total cell volume. We used evacuolated protoplasts in these experiments because they are very stable, fully viable, and easy to work with. Permeabilization in these experiments was achieved by lowering the osmoticum in the medium (Hicks et al., 1996) or by adding low amounts of Triton X-100 (Merkle et al., 1996). In this system, fluorescently labeled NLS substrates were added to the permeabilized protoplasts and import was measured by fluorescence microscopy. Import was rapid and specific for functional NLS substrates, and, in contrast to yeast and vertebrate import systems, NLS protein import can occur in the absence of an exogenously added cytosolic fraction and an ATP-regenerating system. These results suggest that NLS import factors are retained in the permeabilized protoplasts.

Immunolocalization studies using antibodies against an Arabidopsis importin  homolog (At-IMP) show that importin  is retained in the cytoplasm and nucleus in permeabilized cells, even in the presence of detergents (Hicks et al., 1996). In addition, immunofluorescence and biochemical studies demonstrate that importin  is tightly associated with the nucleus and probably the NPC (Smith et al., 1997). Indirect evidence also suggests that Ran is not fully depleted from this system (Merkle et al., 1996). Thus, it is possible that all of the NLS import factors remain associated with the nucleus, and possibly other structures in the cytoplasm, after the protoplasts are permeabilized. Although these observations are unique to plants, it is difficult to test the function of putative plant NLS import factors biochemically because they are not depleted from the permeabilized protoplasts.

Another unique feature of the plant import process is that NLS protein import can occur at 4°C (Hicks et al., 1996; Merkle et al., 1996). It is interesting that import into chloroplasts (Leheny and Theg, 1994) and mitochondria (Knorpp et al., 1994) in higher plants also occurs at 4°C, indicating that plants evolved mechanisms that allow these different import processes to occur at low temperatures.

The energy requirement for import in eukaryotes is probably conserved, because nuclear import can be blocked only by nonhydrolyzable GTP analogs in vertebrates and plants (Hicks et al., 1996; Merkle et al., 1996; Zupan et al., 1996; Görlich, 1997). Ran is necessary for GTP hydrolysis in vertebrates (Görlich, 1997), which strongly indicates a role for Ran in nuclear translocation that is functionally conserved in eukaryotes.

In vertebrates, a subset of NPC proteins is modified with a single O-linked GlcNAc residue (see below; Davis, 1995). Wheat germ agglutinin is a lectin that specifically binds to GlcNAc residues and to glycosylated proteins at the NPC. When wheat germ agglutinin is added to in vitro systems in vertebrates, translocation of nuclear proteins is blocked, probably by steric hindrance. As in vertebrates, wheat germ agglutinin also recognizes glycoproteins at the periphery of nuclei and NPCs in plants (Heese-Peck et al., 1995; Hicks et al., 1996; Merkle et al., 1996). However, unlike the vertebrate system, wheat germ agglutinin does not block the translocation step of NLS protein import in permeabilized protoplasts (Hicks et al., 1996; Merkle et al., 1996). This may be a result of the unique complex sugar modifications found on the glycosylated NPC proteins of plants (Heese-Peck et al., 1995).

In contrast to import reactions in vertebrates and yeast, the addition of crude cytosolic fractions isolated from plant cells inhibits NLS protein import and binding to purified nuclei. Biochemical fractionation of the cytosol demonstrates that this inhibitor is a low-M_r protein (G.R. Hicks and N.V. Raikhel, unpublished data). It is interesting that two regulators of nuclear import in vertebrates and yeast, p10 and a viral protein R (Vpr) from human immunodeficiency virus-1, are low-M_r proteins that can either stimulate or block NLS protein import in vitro in vertebrate import systems (Tachibana et al., 1996; Popov et al., 1998). A p10- or Vpr-like protein could be the low-M_r inhibitor found in plant cytosolic fractions.

	NUCLEAR IMPORT OF NLS PROTEINS REQUIRES BINDING SITES IN THE NPC AND CYTOPLASMIC IMPORTINS

Protein import into the nucleus depends on binding sites at or in the NPC and import factors (proteins) that shuttle between the cytoplasm and the nucleoplasm. Experiments in which isolated nuclei are incubated with nuclear proteins (e.g. transcription factors) allow one to identify the characteristics of the binding sites. Sites located at the NPC and nuclear envelope of plant nuclei specifically and reversibly bind proteins with three different types of NLSs that function in plant cells (Hicks and Raikhel, 1993; Hicks et al., 1995). The identity of the NLS-binding site was characterized biochemically using protein cross-linking; at least four NLS-binding proteins were identified that specifically associate with the bipartite NLS of the maize transcription factor Opaque-2 (Hicks and Raikhel, 1995a). The binding affinity and biochemical properties of the NLS-binding proteins correlate closely with those of the NLS-binding site, indicating that at least one component of NLS recognition is located at the NPC and nuclear envelope in plant cells (Hicks and Raikhel, 1993, 1995a; Hicks et al., 1995).

Nuclear import is mediated by importins, which are cytoplasmic proteins that form a complex with proteins destined for nuclear import. These proteins bind to NLSs. In mammals and yeast, import depends on both importin  and importin . Homologs of importin  have been identified in plants. Immunolocalization of importin  in tobacco protoplasts demonstrates that this receptor is found in the cytoplasm, nucleus, and nuclear envelope, which is consistent with its function as a nuclear-shuttling NLS receptor (Smith et al., 1997).

An Arabidopsis importin  homolog, At-IMP, which recognizes three types of NLSs that function in plant cells (Smith et al., 1997), is present in roots, stems, leaves, and flowers (Hicks et al., 1996). At-IMP may represent a unique class of NLS receptors that have not been identified in vertebrates and yeast. Unlike yeast and vertebrate importin -subunits, At-IMP recognizes NLSs with high affinity, and, even more intriguing, At-IMP can facilitate NLS protein import in the absence of a -subunit in vertebrate import systems (S. Hubner, H.M.S. Smith, N.V. Raikhel, and D.A. Jans, unpublished data). These results indicate that plants may possess a nuclear import pathway exclusively mediated by importin -subunits.

Another Arabidopsis importin  protein, At-KAP, which is 94% identical to At-IMP, was recently found to complement a temperature-sensitive mutation in the yeast importin  protein SRP1, suggesting that plants and yeast share conserved NLS protein import pathways (Ballas and Citovsky, 1997). Recently, four more importin -like proteins were identified in Arabidopsis (Schledz et al., 1998), suggesting that importin  is probably encoded by a small gene family in higher plants.

Unlike At-IMP, the rice importin -subunit recognizes only mono and bipartite NLSs. In addition, this rice -subunit stimulates NLS protein import in conjunction with a mouse importin -subunit in the vertebrate import system. This observation suggests that a conserved NLS protein import pathway mediated by the / heterodimer also exists in plants (Jiang et al., 1998). More importantly, characterization of At-IMP suggests that plants possess an importin -independent import pathway. Further characterization of import factors in plants is required to define these NLS protein import pathways in plants.

	NUCLEAR IMPORT REQUIRES Ran GTPases AND GTP

Many cellular processes including nuclear import depend on the energy provided by GTP. The GTPase that mediates nuclear import is called Ran. Ran homologs that are approximately 75% identical to other Ran homologs found in vertebrates and fungi have been identified in Arabidopsis (Haizel et al., 1997), tomato (Ach and Gruissem, 1994), tobacco (Merkle et al., 1994), fava bean (Saalbach and Christov, 1994), and Lotus japonicus (Borg et al., 1997). In plants Ran appears to be encoded by a small gene family whose members are expressed ubiquitously in various organs (Ach and Gruissem, 1994; Merkle et al., 1994; Haizel et al., 1997). As in mammals and yeast, Ran localizes to the nucleus in plant cells (Ach and Gruissem, 1994; Merkle et al., 1994). A mutation in the Schizosaccharomyces pombe Ran gene pim1 has a cell-cycle defect that causes chromosomes to condense during DNA replication. Overexpression of Ran cDNAs from tomato or tobacco suppresses the pim1 phenotype, indicating that these Ran proteins have a function in plants similar to that in yeast (Ach and Gruissem, 1994; Merkle et al., 1994). However, a direct role of plant Ran-like proteins in nuclear import and export is yet to be described.

In mammals and yeast, the proteins that stimulate RanGTPase activity, RanGAP1 and RanBP1, are found exclusively in the cytoplasm, whereas RCC1, which converts RanGDP to RanGTP, is found exclusively in the nucleus (Görlich, 1997). The localization of these Ran-activating and exchange proteins probably creates a steep RanGTP gradient across the nuclear envelope that may be essential for nuclear import and export (Görlich, 1997; Nigg, 1997).

Recently it was shown by the yeast two-hybrid assay that an Arabidopsis Ran homolog, At-Ran1, interacts with two homologous RanBP1 proteins, At-RanBP1a and At-RanBP1b. In addition, biochemical studies show that these Ran-activating proteins interact with all three isoforms of At-Ran. Similar to the expression patterns of the At-Ran genes, At-RanBP1a and At-RanBP1b are expressed ubiquitously in Arabidopsis plants (Haizel et al., 1997). However, it is not known whether At-RanBP1a and At-RanBP1b can stimulate RanGTPase activity in conjunction with a plant RanGAP1 protein. In addition, localization studies should determine whether At-RanBP1a and At-RanBP1b are localized to the cytoplasm in plant cells.

	REGULATION OF NUCLEAR IMPORT

NLS protein import is not always a constitutive process; in fact, regulated nuclear import of various types of signaling proteins, including transcription factors and protein kinases, has been observed in all eukaryotes, including plants (for reviews, see Hicks and Raikhel, 1995b; Jans and Hubner, 1996; Nagatani, 1998). These classes of signaling proteins are linked to signal transduction pathways, where the transcription factor or kinase translocates into the nucleus in response to the appropriate environmental or chemical stimulus. After protein synthesis, regulation is achieved by anchoring the nuclear-signaling protein to cell structures (i.e. cytoskeleton or ER) or by inhibiting the interaction of importin  with the NLS. These mechanisms allow the cell to tightly control the activity of these signaling proteins. Thus, regulating NLS protein import is the cell's method of controlling gene expression (for reviews, see Hicks and Raikhel, 1995b; Jans and Hubner, 1996). Regulated nuclear targeting in plants is a relatively small field so far, and it was recently reviewed (Hicks and Raikhel, 1995b; Nagatani, 1998), so it will not be covered here in detail. In addition, studies suggest that the import apparatus itself may be regulated, which would allow the cell to control the expression of the cell genome globally (for review, see Jans and Hubner, 1996). Protein is imported to the nucleus continuously, so importins are needed all the time. Nevertheless, a gene encoding importin  was found to be up-regulated by light in rice (Shoji et al., 1998). Perhaps this represents a response to the need for greatly accelerated import of nuclear proteins during greening or in the diurnal cycle.

	NUCLEOPORINS ARE PROTEIN COMPONENTS OF THE NPC

Although the structure of the NPC is well characterized, its role in translocation is not fully understood. The identification and characterization of NPC proteins, called nucleoporins, is a crucial step toward understanding the mechanisms involved in the translocation of proteins and ribonucleoproteins through the NPC. In vertebrates and yeast only a fraction of the approximately 100 different nucleoporins has been identified and characterized.

Study of the NPC in plants has been limited to biochemical characterization. Antibodies raised against the yeast nucleoporin Nsp1 recognize a 100-kD protein in nuclear matrix preparations isolated from carrot suspension cells, suggesting that an Nsp1-like protein is an element of the NPC in plants (Scofield et al., 1992). Electron microscopy demonstrated that the NLS-binding site associated with purified plant nuclei is a component of the NPC (Hicks and Raikhel, 1993; Hicks et al., 1995). In addition, immunofluorescence and biochemical observations indicate that importin  is associated with the NPC, and this receptor is probably a component of the NLS-binding site in plants (Smith et al., 1997).

A subset of nucleoporins modified with a terminal GlcNAc residue is located at the NPC in vertebrates and plants (Davis, 1995; Heese-Peck et al., 1995). It is interesting that none of the yeast nucleoporins are modified by GlcNAc residues (Davis, 1995). In vertebrates, single GlcNAc residues are attached to this subset of nucleoporins (Davis, 1995), whereas the plant nucleoporins are modified with larger polysaccharides that contain at least five GlcNAc residues (Heese-Peck et al., 1995). Although the significance of the GlcNAc residues is not known, the presence of sugar residues has been useful in the purification and identification of these nucleoporins from vertebrate cells (Davis, 1995). Several of these glycosylated nucleoporins interact with NLS import factors in vitro, indicating that they are active in nuclear import (Görlich, 1997).

Recently, several glycoproteins were purified by lectin-affinity chromatography from tobacco nuclear extracts, and one corresponding gene has been cloned. This gene encodes a 40-kD glycoprotein called "gp40," which displays 28% to 34% amino acid identity to aldose-1-epimerases from bacteria. Antibodies raised against gp40 demonstrate that it is localized to the periphery of the nucleus in fixed tobacco protoplasts; the biochemical characterization of gp40 from isolated nuclei strongly suggests that it is a component of the NPC in plants (Heese-Peck and Raikhel, 1998). Bacterial aldose-1-epimerases function in carbohydrate metabolism, but we do not know if gp40 has a similar enzyme activity.

The gene encoding O-GlcNAc transferase, the enzyme that mediates the attachment of O-GlcNAc to proteins, has been isolated recently from vertebrates (Kreppel et al., 1997; Lubas et al., 1997). Sequence analysis shows it to have extensive similarity to a protein encoded by an Arabidopsis gene known as SPINDLY (Jacobsen et al., 1996). The SPINDLY gene was originally isolated as a GA-responsive mutant, indicating involvement in GA signal transduction. The gp40 protein has been isolated based on its sugar moiety; with its terminal GlcNAc, it is a useful tool with which to evaluate the potential O-GlcNAc transferase activity of SPINDLY.

	IMPORTIN  INTERACTS WITH THE CYTOSKELETON IN PLANTS

Although many import receptors have been identified in vertebrates and yeast, the transport mechanism that targets these import complexes from the cytoplasm to the NPC is unknown. Intracellular transport of organelles (Hirokawa, 1998; Mermall et al., 1998), viruses (Greber et al., 1997; Sodeik et al., 1997), and mRNA protein complexes (Hovland et al., 1996; Bassel and Singer, 1997) are mediated by the cytoskeleton. A fundamental question in nuclear transport is how cytoplasmically synthesized proteins are targeted and directed to the NPC. The cytoskeleton could play such a role by mediating the transport of NLS-containing proteins from the cytoplasm to the NPC prior to protein import.

Several observations suggest that importin  associates with the cytoskeleton in plants. Immunolocalization of At-IMP in tobacco protoplasts has displayed radial cytoplasmic staining extending from the nucleus to the plasma membrane in a cytoskeleton-like pattern (Fig. 2b; Smith et al., 1997). At-IMP is not depleted from tobacco protoplasts after permeabilization, indicating that it is associated with structures in the cytoplasm and nucleus (Hicks et al., 1996). Importin  contains highly hydrophobic armadillo repeats of 42 amino acids in length; these are implicated in protein-protein interaction (Hicks et al., 1996). Thus, importin  could interact with the cytoskeleton, because other proteins containing armadillo repeats have such interactions with these structures (Barth et al., 1997).

View larger version (35K): [in this window] [in a new window]   Figure 2. Importin  colocalizes with microtubules in the cytoplasm of tobacco protoplasts. Most of the cytoplasmic importin  (b) colocalized with microtubules (a) in fixed tobacco protoplasts. The green image displays the microtubules and the red image displays the cytoplasmic strands of importin . Superimposing these two images (c) demonstrates coalignment, shown in yellow/orange.

We recently demonstrated that importin  colocalizes with microtubules and microfilaments by double-immunofluorescence, confocal laser-scanning microscopy in tobacco protoplasts (Fig. 2; Smith and Raikhel, 1998). Depolymerization of the cytoskeleton disrupts the cytoskeleton-like pattern of At-IMP in the cytoplasm. It is interesting that when the microtubules are depolymerized importin  staining in the cytoplasm is diffuse. However, depolymerization of microfilaments causes At-IMP to accumulate inside the nucleus, indicating that the microfilaments are involved in retaining this receptor in the cytoplasm (Smith and Raikhel, 1998).

In vitro cytoskeleton-binding assays demonstrate that At-IMP associates with microtubules and microfilaments in an NLS-dependent manner. This association could be essential for the transport of importin /NLS-containing protein complexes in the cytoplasm. The NLS-dependent association of importin  with the cytoskeleton could also be important for anchoring the NLS receptors in the cytoplasm, which could be a mechanism for regulating the expression of newly synthesized NLS-containing proteins.

	A WORKING MODEL FOR TRANSPORT FROM THE CYTOPLASM TO THE NPC

From these observations and the results of studies using other systems, we can develop a working model for the role of the cytoskeleton in NLS protein transport. The model in Figure 3, for example, shows that microfilaments could serve as sites to assemble importin  with NLS-containing proteins. The yeast importin -subunit Srp1p binds directly to the actin-related protein Act2p, which has a distribution similar to that of the microfilaments in the cytoplasm of yeast cells (Yan et al., 1997); therefore, it is tempting to speculate that Act2p could be involved in retaining importin  in the cytoplasm. This retention mechanism may perform as an assembler of the importin /NLS-containing protein complexes, because the translation of many mRNAs that encode nuclear proteins occurs on microfilaments (Hovland et al., 1996; Bassel and Singer, 1997). In our model, as the NLS proteins are synthesized and folded, they are assembled with importin , forming transport complexes that are then loaded onto microtubule tracks for transport to the NPC.

View larger version (19K): [in this window] [in a new window]   Figure 3. A schematic model for intracellular transport of the importin/NLS-containing protein complex. In this hypothetical model, importin  forms transport-competent complexes on microfilaments where nuclear proteins are synthesized. Protein "X," which anchors importin  to the microfilaments, could be Act2-like protein, which interacts with importin . In the next step, assembled complexes are loaded onto microtubules for transport to the NPC. The transport mechanism could be mediated by a microtubule motor protein (M). Thus, this transport step would precede the binding and translocation steps of import.

The model is supported by observations made in neurons. Transport of NLS-containing proteins in neurons was analyzed by microinjecting fluorescently labeled NLS substrates into the termini of neurons and monitoring transport along the axon. The movement was unidirectional toward the nucleus and dependent on functional NLSs. The depolymerization of microtubules before microinjection blocked NLS transport along the axon. Therefore, it was concluded that transport of proteins toward the nucleus in neurons is microtubule dependent (Ambron et al., 1992). Transport is probably facilitated by a microtubule motor protein because importin  does not share similarity to known motor proteins such as kinesin or dynein.

Future work should be directed toward understanding mechanistic details of import in plants. For example, is the importin -independent import pathway a unique feature of plants and does it define a novel pathway for import? Another exciting area of investigation will be the molecular mechanisms involved in intracellular transport of importin /NLS protein complexes in the cytoplasm. An NLS-protein transport system should be developed to characterize the movement of NLS-containing proteins along microtubules. The identification of cytoskeleton-binding factors that mediate importin  interaction with microtubules and microfilaments should also provide interesting new details of protein targeting to NPCs. The development of a cytoskeleton transport system could ultimately test the function of these binding proteins in the intracellular transport of NLS-containing proteins. The study of import in plants has uncovered some important new details and is now poised to broaden our knowledge of this process in more fundamental ways.

	FOOTNOTES

   Received December 9, 1998; accepted January 21, 1999.

	ABBREVIATIONS

Abbreviations: NLS, nuclear localization signal. NPC, nuclear pore complex.

	ACKNOWLEDGMENT

We thank Dr. Glenn Hicks for critical reading of the manuscript.

	LITERATURE  CITED

Top Introduction References

Ach RA, Gruissem W (1994) A small GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant. Proc Natl Acad Sci USA 91: 5863-5867 [Abstract/Free Full Text]

Ambron RT, Schmied R, Huang CC, Smedman M (1992) A signal sequence mediates the retrograde transport of proteins from the axon periphery to the cell body and then into the nucleus. J Neurosci 12: 2813-2818 [Abstract]

Ballas N, Citovsky V (1997) Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein. Proc Natl Acad Sci USA 94: 10723-10728 [Abstract/Free Full Text]

Barth AI, Nathke IS, Nelson WJ (1997) Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways. Curr Opin Cell Biol 9: 683-690 [CrossRef][ISI][Medline]

Bassel G, Singer RH (1997) mRNA and cytoskeletal filaments. Curr Opin Cell Biol 9: 109-115 [CrossRef][ISI][Medline]

Borg S, Brandstrup B, Jenson TJ, Poulsen C (1997) Identification of new protein species among 33 different small GTP-binding proteins encoded by cDNAs from Lotus japonicus, and expression of corresponding mRNAs in developing root nodules. Plant J 11: 237-250 [CrossRef][ISI][Medline]

Davis L (1995) The nuclear pore complex. Annu Rev Biochem 64: 865-896 [CrossRef][ISI][Medline]

Görlich D (1997) Nuclear protein import. Curr Opin Cell Biol 9: 412-419 [CrossRef][ISI][Medline]

Greber UF, Suomalainen M, Stidwill RP, Boucke K, Ebersold W, Helenius A (1997) The role of the nuclear pore complex in adenovirus DNA entry. EMBO J 16: 5998-6007 [CrossRef][ISI][Medline]

Haizel T, Merkle T, Pay A, Fejes E, Nagy F (1997) Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis. Plant J 11: 93-103 [CrossRef][ISI][Medline]

Harter K, Kircher S, Frohnmeyer H, Krenz M, Nagy F, Schafer E (1994) Light-regulated modification and nuclear translocation of cytosolic G-box binding factors in parsley. Plant Cell 6: 545-559 [Abstract]

Heese-Peck A, Cole RN, Borkhsenious ON, Hart GW, Raikhel NV (1995) Nuclear pore complex proteins from higher plants are modified by novel O-linked oligosaccharides. Plant Cell 7: 1459-1471 [Abstract]

Heese-Peck A, Raikhel NV (1998) A glycoprotein modified with terminal N-acetylglucosamine and localized at the nuclear rim shows sequence similarity to aldose-1-epimerase. Plant Cell 10: 599-612 [Abstract/Free Full Text]

Hicks GR, Raikhel NV (1993) Specific binding of nuclear localization sequences to plant nuclei. Plant Cell 5: 983-994 [Abstract/Free Full Text]

Hicks GR, Raikhel NV (1995a) Nuclear localization signal binding proteins in higher plant nuclei. Proc Natl Acad Sci USA 92: 734-738 [Abstract/Free Full Text]

Hicks GR, Raikhel NV (1995b) Protein import into the nucleus: an integrated view. Annu Rev Cell Dev Biol 111: 155-188 [CrossRef]

Hicks GR, Smith HMS, Lobreaux S, Raikhel NV (1996) Nuclear import in permeabilized protoplasts from higher plants has unique features. Plant Cell 8: 1337-1352 [Abstract]

Hicks GR, Smith HMS, Shieh M, Raikhel NV (1995) Three classes of nuclear import signals bind to plant nuclei. Plant Physiol 107: 1055-1058 [Abstract]

Hirokawa N (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279: 519-526 [Abstract/Free Full Text]

Hovland R, Hesketh JE, Pryme IF (1996) The compartmentalization of protein synthesis: importance of cytoskeleton and role in mRNA targeting. Int J Biochem Cell Biol 28: 1089-1105 [CrossRef][Medline]

Jacobsen SE, Binkowski KA, Olsewski NE (1996) SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis. Proc Natl Acad Sci USA 93: 9292-9296 [Abstract/Free Full Text]

Jans DA, Hubner S (1996) Regulation of protein transport to the nucleus: central role in phosphorylation. Physiol Rev 76: 651-685 [Abstract/Free Full Text]

Jiang C-J, Imamoto N, Matsuki R, Yoneda Y, Yamamoto N (1998) Functional characterization of a plant importin  homologue. J Biol Chem 273: 24083-24087 [Abstract/Free Full Text]

Knorpp C, Hugosson M, Sijoling S, Eriksson AC, Glaser E (1994) Tissue-specific differences of the mitochondrial protein import machinery: in vitro import, processing and degradation of the pre-fb subunit of the ATP synthase in spinach leaf and root mitochondria. Plant Mol Biol 26: 571-579 [Medline]

Kreppel LK, Blomberg MA, Hart GW (1997) Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats. J Biol Chem 272: 9308-9315 [Abstract/Free Full Text]

Leheny EA, Theg SM (1994) Apparent inhibition of chloroplast protein import by cold temperature is due to energetic considerations, not membrane fluidity. Plant Cell 6: 427-437 [Abstract]

Lubas WS, Frank DW, Krause M, Hanover JA (1997) O-linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats. J Biol Chem 272: 9316-9324 [Abstract/Free Full Text]

Merkle T, Haizel T, Matsumoto T, Harter K, Dallmann G, Nagy F (1994) Phenotype of the fission yeast cell cycle regulatory mutant pim1-46 is suppressed by a tobacco cDNA encoding a small, Ran-like GTP-binding protein. Plant J 6: 555-565 [CrossRef][ISI][Medline]

Merkle T, Leclerc D, Marshallsay C, Nagy F (1996) A plant in vitro system for the nuclear import of proteins. Plant J 10: 1177-1186 [CrossRef][ISI][Medline]

Mermall V, Post PL, Mooseker MS (1998) Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science 279: 527-533 [Abstract/Free Full Text]

Nagatani A (1998) Regulated nuclear targeting. Curr Opin Plant Biol 1: 470-474 [CrossRef][Medline]

Nigg EA (1997) Nucleocytoplasmic transport: signals, mechanism and regulation. Nature 386: 779-787 [CrossRef][Medline]

Popov S, Rexach M, Zybarth G, Reiling N, Lee MA, Ratner L, Lane CM, Moore MS, Blobel G, Bukrinsky M (1998) Viral protein R regulates nuclear import of the HIV-1 pre-integration complex. EMBO J 17: 909-917 [CrossRef][ISI][Medline]

Saalbach G, Christov V (1994) Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein. Plant Mol Biol 24: 969-972 [CrossRef][ISI][Medline]

Schledz M, Leclerc D, Neuhaus G, Merkle T (1998) Characterization of four cDNAs encoding different importin alpha homologs from Arabidopsis. Plant Physiol 116: 868

Scofield GN, Beven AF, Shaw PJ, Doonan JH (1992) Identification and localization of a nucleoporin-like protein component of the plant nuclear matrix. Planta 187: 414-420

Shoji K, Iwasaki T, Matsuki R, Miiyao M, Yamamoto N (1998) Cloning of a cDNA encoding an importin-alpha and down-regulation of the gene by light in rice leaves. Gene 212: 279-286 [CrossRef][ISI][Medline]

Smith HMS, Hicks GR, Raikhel NV (1997) Importin  from Arabidopsis thaliana is a nuclear import receptor that recognizes three classes of import signals. Plant Physiol 114: 411-417 [Abstract]

Smith HMS, Raikhel NV (1998) Nuclear localization signal receptor importin  associates with the cytoskeleton. Plant Cell 10: 1791-1799 [Abstract/Free Full Text]

Sodeik B, Ebersold MW, Helenius A (1997) Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol 136: 1007-1021 [Abstract/Free Full Text]

Tachibana T, Hieda M, Sekimoto T, Yoneda Y (1996) Exogenously injected nuclear import factor p10/NTF2 inhibits signal-mediated nuclear import and export of proteins in living cells. FEBS Lett 397: 177-182 [CrossRef][ISI][Medline]

Yan C, Leibowitz N, Melese T (1997) A role for the divergent actin gene, ACT2, in nuclear pore structure and function. EMBO J 16: 3572-3586 [CrossRef][ISI][Medline]

Zupan JR, Citovsky V, Zambryski P (1996) Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells. Proc Natl Acad Sci USA 93: 2392-2397 [Abstract/Free Full Text]

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