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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Nuclear Import of Plant Proteins

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Introduction

Overview

In this Chapter, we will focus primarily on protein import into the nucleus of plants. As in other eukaryotes the partitioning of genetic information into the nucleus necessitates the import and export of macromolecules such as proteins, nucleic acids and protein/nucleic acid complexes across the nuclear envelope. These transport processes are essential and are subject to stringent regulatory controls. In plants, it is clear that in addition to the maintenance of basic cellular processes, the regulated import of proteins plays a vital role in development. As we will discuss, the nuclear import of proteins in a selective manner is essential in responses of plants to light and results in the dramatic morphological changes that occur as plants switch from growth in the dark to growth in the light.1 Protein translocation across the nuclear envelope is also a process that is utilized by pathogenic viruses2 and tumor-inducing bacteria in the genus Agrobacterium3 to transport protein/nucleic acid complexes into the nucleus for replication and even incorporation of pathogen DNA into the host genome.

The import pathway for proteins, themselves key factors regulating nuclear transport processes, is the best understood of the nuclear transport processes in plants. Numerous import signals have been characterized, and we are beginning to identify and understand the major components of the import machinery. As expected many of these factors are conserved between plants, animals and fungi, but there are surprising results indicating subtle differences in preferences for nuclear localization signals (NLSs),3,4 the mechanics of import receptor function5, and potential plant-specific import factors.6 As we move forward plants are contributing new knowledge such as potential mechanisms for the targeting of proteins to the nuclear envelope and nuclear pore complex (NPC).7 Recent efforts have begun to focus on other nuclear transport processes in plants such as the export of proteins and nucleic acids.8,9

Protein Import in Animals and Yeast

Our knowledge of nuclear transport in vertebrates and yeast is more advanced than our understanding in plants. Among the reasons for this historically are: 1. the ease of recovering nuclei from Xenopus oocytes that are intact for morphological studies, 2. the development of in vitro systems in Xenopus for reconstituting the assembly of nuclei and NPCs, 3. the reconstitution of cytosol-dependent nuclear import in permeabilized mammalian cells, 4. the rapid genetics possible in yeast, 5. the large biomedical research community and potential importance of nuclear processes for medicine. However as we are discovering nuclear translocation is a critical aspect of plant growth and development and thus has broad implications for agriculture in terms of disease and stress resistance and other crop improvements, which are the keys to feeding an expanding worldwide population. To place our knowledge of plant nuclear import in perspective, we will overview processes in animals and yeast throughout the Chapter highlighting important advances and unique aspects in plants. Import from the perspective of non-plant systems is covered in greater detail in other Chapters. There are also excellent recent reviews that are focused on the nuclear/cytoplasmic transport of proteins, nucleic acids and their complexes4,10–22as well as the structure and function of the NPCs in vertebrates and yeast.23–30

Nuclear Translocation in Plants

Nuclear Pore Complex

The channels through which all transport substrates must pass are the NPCs which are macromolecular complexes embedded in the double-membrane nuclear envelope. The NPCs are estimated to have a mass of 125 MDa in higher eukaryotes and to be composed of 50 to 100 different proteins collectively known as nucleoporins. Morphologically, an NPC is composed of a nucleoplasmic and a cytoplasmic ring. Eight spokes are found within the rings that extend toward a central channel resulting in eight 9 nm channels thought to function in the diffusion of small molecules across the nuclear envelope. A basket-like structure extends from the nucleoplasmic face and fibrils have been observed extending from the cytoplasmic face. Thus far, fewer than 20 nucleoporins have been purified from vertebrates. The NPCs in yeast are less complex (mass about 66 MDa) and are not dissociated and reassembled during mitosis as in higher eukaryotes; nevertheless the development of methods to purify intact complexes31 has permitted the identification and sequencing of all 30 of its nucleoporins. Even with such information available understanding the assembly and function of the NPC will be a daunting task.32

A number of vertebrate NPC proteins have been implicated in nuclear import.30 They include Nup358 which is located on the cytoplasmic filaments. Nup 358 has multiple Ran binding sites and binds to the protein transporter importin β (See Components and Mechanisms of Protein Import) via FXFG (single amino acid code where X represents an amino acid with a small or polar side chain) repeats33 which are characteristic of many nucleoporins. Other nucleoporins reported to bind to importin β include Nup153,34 Nup214,35 Nup116p, Nup100p,36 and p62.37 The p62 protein was one of the first nucleoporins to be purified, and like many nucleoporins from vertebrates it is modified by single O-linked N-acetylglucosamine (O-GlcNAc) residues. While the O-GlcNAc is probably not essential for import, the binding of the lectin wheat germ agglutinin (WGA) inhibits nuclear import in vertebrates and has been used for the identification and purification of nucleoporins from higher eukaryotes (For review see ref. 38).

From electron microscopy studies beginning in the 1970s we know that nuclear pores in plants are morphologically similar to those of other organisms.39 However there have been few reports in which plant nucleoporins have been purified (For review see ref. 40). Scofield et al (ref. 41) localized a protein at the NPC and identified a 100 kDa polypeptide in a nuclear matrix fraction from carrots using an antibody to the yeast nucleoporin NSP100; however successful purification was not reported. Other studies indicate that nuclear envelope fractions from maize and tobacco nuclei contain a subset of proteins in the NPC fraction that can bind NLSs specifically.42–44 The binding site is at the NPC indicating a role for plant nucleoporins in protein import.42 This finding is consistent with the unusually tight association of at least one plant importin α NLS receptor with the nuclear envelope in purified nuclei and intact cells.45,46

Using WGA as a probe it is clear that GlcNAc-modifications are present at the periphery of the nucleus,47–49 and electron microscopy has shown that some of these modifications are present at the NPC.47 In fact biochemical characterization of tobacco nuclear fractions has shown that the glycans are attached to proteins via an O-linkage and the moities are longer than five sugar residues in length ending with a terminal GlcNAc residue. This is a novel modification not found in vertebrates, which contain only a single O-GlcNAc residue.47 Although the functional significance of O-GlcNAc modifications is not clear the modification can be used to advantage for purifying plant NPC proteins. Using lectin affinity chromatography four O-GlcNAc proteins were purified from nuclear envelope fractions of cultured tobacco cells. Peptide sequence was obtained from a protein of 40 kDa (gp40) that shares about 30% identity with aldose-1-epimerases (also known as mutarotases) which are involved in aldose sugar metabolism in bacteria.38 Their role in higher eukaryotes is not well defined but they do share structural similarities to the glucose carrier from erythrocytes. Interestingly, it has been reported that glycosylated proteins can be imported in a sugar-specific and NLS-independent manner in mammalian cells in vitro.50 Thus one speculation is that gp40 may be involved in such an import system in plants by binding to glycosylated proteins destined for import.40 Regardless of the role eventually assigned to gp40 a procedure to purify NPC proteins from plants should permit additional proteins to be characterized. The availability of Arabidopsis genome sequence should also contribute to the identification of plant nucleoporins, although it should be noted that beyond several very short repeat motifs such as FXFG there are few similarities even between vertebrate and yeast nucleoporins. While interesting in itself, it suggests that the identification of plant nucleoporins based on protein identity across kingdoms will be only partially successful.

Import Signals in Plants

Several types of transport can occur through NPCs. Ions and small proteins that are typically less than 20 to 30 kDa can pass by simple diffusion through the 9 nm channel. However even small macromolecules such as histones (14 kDa)51 or tRNAs52 cross the NPC via active processes permitting their translocation to be under cellular control. There is evidence that even calcium ions may be subject to selective concentration within the nucleus;53 this may relate to the suggestion that calcium plays a role in the regulation of active import.54 Nuclear transport processes are mediated by specific import receptors that recognize signals located within their respective substrates. In the case of protein import, NLSs interact with the import machinery that facilitates translocation through the NCP. Unlike most signals for organelles including the chloroplasts, mitochondria, vacuoles, peroxisomes and endoplasmic reticulum, NLSs are not proteolytically removed following import. This permits NLS-containing proteins to shuttle in and out of the nucleus and to be re-imported following post-mitotic nuclear assembly. Many transcription factors and cell cycle-regulatory proteins are able to exert their activities upon the cell based on their relative abundances in the cytosol compared to the nuceloplasm.10,54 Most NLSs are classified as either monopartite or bipartite. The classic monopartite NLS is from the SV40 large T-antigen and is composed of a single short region enriched in the basic residues arginine and lysine, whereas the nucleoplasmin NLS, which first defined the bipartite class, is composed of two basic domains separated by a spacer of variable length and composition. There are also NLSs that are less typical. One unusual NLS class is typified by the signal within the Matα2 protein which requires both basic and hydrophobic residues (for review see ref. 44).

As in other organisms nuclear localization signals in plants cannot be defined by a strict consensus sequence and regions of basic amino acids are common within proteins, particularly regions involved in DNA binding. Thus, putative NLSs must be examined for activity in vivo to confirm their function. A number of NLSs have been carefully defined in such a manner in plants. These signals include at least one member for each of the three NLS classes. As examples, the transcriptional activitor R from maize possesses three functional NLSs, two SV40-like (one of them, NLS M, is defined as MSERKRREKL) and one Matα2-like signal (NLS C is defined as MISEALRKAIGKR), each of which are sufficient to target a reporter protein to the nucleus in vivo. Another transcription factor from maize, Opaque-2 possesses two signals, one SV40-like and one bipartite signal (NLS B is defined as RKRKESNRESARRSRYRK), that are sufficient to target a reporter protein to the nucleus. For R and Opaque2, mutations within the intact proteins indicate that multiple NLSs are necessary for efficient targeting in vivo suggesting cooperativity among NLSs for import which is also true in vertebrates.4

It is generally accepted that most NLSs can function across kingdoms pointing to a high degree of functional conservation. For example the SV40 large T-antigen NLS functions in plants (See for example refs. 55, 56) and the single bipartite NLS from the VirD2 protein of the plant pathogen Agrobacterium functions in plant, Xenopus, Drosophila, mammalian, and yeast cells.57–59 Agrobacterium is an opportunistic pathogen that infects a wide variety of plant species.3 In the coarse of pathogenesis Agrobacterium interacts with the host cell and transfers pathogen DNA (T-DNA) into the host cell nucleus through the NPC via the cooperative action of the VirD2 and VirE2 proteins. The T-DNA integrates into the plant genome and utilizes host factors to transcribe pathogen sequences. Agrobacterium has been a valuable system for studying import and will be discussed in later sections.

Plant import is not strictly conserved when compared to import in other kingdoms however. The yeast Matα2 NLS targets a β-glucuronidase (GUS) reporter protein to the nucleus in onion epidermal cells which is consistent with the specific association of this class of NLSs with an import receptor from Arabidopsis.44–46 Interestingly, the Mata2 signal does not function in mammals60,61 although other yeast NLSs are known to function in vertebrates.62 Another exception is from Agrobacterium. As mentioned the NLS from VirD2 is broadly functional across kingdoms. Fascinatingly, VirE2 which contains two functional bipartite signals does not function in any of the non-plant organisms described for VirD2.57,3,59 However a single amino acid change which alters one NLS to conform to the animal bipartite consensus permits the NLS to function in Xenopus and Drosophila.57 Overall these results indicate that there may be subsets of import receptors or other components in plants that are not present in animals and fungi.

Components and Mechanisms of Protein Import

The key components of the classical NLS protein import pathway have been identified within the past decade and are the NLS-receptor importin α, the broad specificity transporter importin β, the GTPase Ran, and the Ran-interacting factor NTF2.21,30,63 In the first event of the protein import pathway, NLSs within nuclear proteins are recognized and bound by the NLS receptor importin α in the cytoplasm (Fig. 1). Another factor, importin β interacts with importin α (via an importin β binding domain within importin α) completing the trimeric import complex. It is importin β that then interacts with specific proteins of the NPC facilitating translocation through the NPC. Thus for protein import importin α functions as an adapter that recognizes the NLS and associates with importin β, whereas importin β functions as the actual transporter. The directionality of import is determined by the binding of the small ras-related GTPase Ran to importin β. Following import of the trimeric complex, the GTP-bound form of Ran (Ran-GTP) binds to importin β in the nucleoplasm resulting in the release of importin α and the NLS-containing cargo from the complex. The importin β/Ran-GTP heterodimer is then exported to the cytoplasm where Ran-GTP is hydrolyzed to Ran-GDP leading to the release of importin β for subsequent rounds of import via reassociation with importin α and NLS-containing cargo. Since monomeric importin α does not typically interact directly with the NPC it is exported back to the cytoplasm via its own export receptor, CAS,64 for subsequent rounds of import.

Figure 1. Schematic overview of nuclear protein import.

Figure 1

Schematic overview of nuclear protein import. Import of NLS-containing proteins is dependent upon the formation of a trimeric import complex in the cytoplasm. Following translocation, binding of Ran-GTP to importin β releases importin α (more...)

The energy and directionality of import are hypothesized to depend on the enrichment of Ran-GTP in the nucleoplasm compared to the cytoplasm which contains mostly Ran-GDP. This is accomplished through the action of the nucleotide exchange factor RCC1 in the nucleus that enhances nucleotide exchange favoring Ran-GTP and the GTPase-activating protein RanGAP1 that favors conversion to Ran-GDP in the cytoplasm. The GTPase activity is further stimulated by Ran-BP1. Import is effectively a process of facilitated diffusion utilizing a gradient of Ran-GTP; a loose analogy would be to envision the import apparatus as an antiporter that causes accumulation of protein against a gradient of Ran-GTP. In the cytoplasm, the factor NTF2 interacts with Ran-GDP and NPC proteins and functions as a receptor/transporter for the re-import of Ran preventing its depletion from the nucleoplasm.65,66 It should be noted that Ran plays essential roles beyond nuclear trafficking such as regulation of cell cycle progression.63

The selectivity and range of cargoes shuttled across the NPC is determined by different isoforms of importin α and importin β within the cell. The importin α receptor for NLS-mediated protein import has been found either as a single gene (SRP1) in yeast or as a small gene family in other organisms. In vertebrates at least six genes encoding importin α isoforms has been reported.21 There is also evidence of distinct but overlapping preferences for different NLSs and possible cell-specific roles for the different importin αs.57,67–71 Conversely, the range of importin β-like proteins, many of which are only distantly related, is far more diverse. This is due to the fact that members of the importin β family participate not only in the import of NLS-containing proteins but also function directly as import (importins) and export (exportins) receptor/transporters for other essential cargoes via their interaction with the NPC. Some examples include transportin 1 (import of hnRNP proteins), CRM1 (exportin 1; export of proteins containing a nuclear export signal), exportin-t (export of tRNAs), importin 7 (import of ribosomal proteins), and snurportin 1 (import of U snRNPs) (For review see ref. 21). The recent understanding of this receptor/transporter family has provided mechanistic details about the transport of diverse cargoes and highlighted potential ways in which the translocation of essential macromolecules is coordinated during the cell cycle.

In higher plants we have only recently begun to identify components of the import apparatus. Hicks et al (ref. 48) identified a homologue of the importin α receptor, At-IMP α from Arabidopsis. Immunologically related proteins are found in all organs examined including roots, stems, leaves, and flowers as expected for an essential factor. There is also evidence that At-IMP α is phosphylated in vitro in the presence of cytosolic extracts suggesting a potential mechanism for controlling NLS binding or interaction with other proteins (Hicks and Raikhel unpublished). At the cellular level, At-IMP α is localized in the cytoplasm and nucleoplasm and at the nuclear envelope as expected for a receptor that shuttles between compartments. One unusual aspect of At-IMP α is its tight association with the nuclear envelope even in plant cells that have been treated to permeabilize the plasma membrane and deplete cytosolic contents;48 in animal cells endogenous importin α is mostly cytosolic and easily depleted from permeabilized cells. The plant receptor binds specifically in vitro to monopartite, bipartite and Matα2-like NLSs indicating broad NLS selectivity.5,45 Unlike yeast and mammalian orthologs that require importin β for high-affinity binding to NLSs, At-IMP α binds with high affinity (Kd of 5 to10 nM) in the absence of an importin β subunit, although At-IMP α is capable of binding to mouse importin β. This is consistent with the finding that At-IMP α can mediate association of import substrate at the nuclear envelope in permeabilized animal cells, whereas mouse importin α absolutely requires importin β.5 In fact, At-IMP α can mediate nuclear protein import in permeabilized animal cells in the absence of exogenous importin β indicating that At-IMP α shares some properties with importin β.5 This is a surprising result given that At-IMPa shares significant homology with other importin αs which require importin β and indicates the possibility of an importin β-independent pathway in plants.

Although At-IMP α has some unusual properties, other plant importin α homologues are more typical of their animal and yeast counterparts. Using the Agrobacterium protein VirD2 as bait in a yeast two-hybrid screen of an Arabidopsis library, Ballas and Citovsky72 identified an importin α homologue (AtKAP α) that has high identity with At-IMP α except for a 64 amino acid extension at the amino-terminus. AtKAP α interacts specifically with the carboxy-terminal NLS of VirD2 both in vitro and in vivo in the two-hybrid assay, and the AtKAP α gene was found to complement a temperature-sensitive yeast mutant srp1–31 (yeast importin α). Cytosolic extract containing AtKAP α protein was able to restore import to cells of srp1–31 in an in vitro import system using permeabilized yeast cells in which import is dependent upon exogenous cytosol. Interestingly, VirE2 does not interact with AtKAP α suggesting an alternative pathway for its import (For discussion see ref. 72). A two-hybrid screen has identified a candidate for a VirE2 import protein (VIP1) that is related to basic leucine zipper proteins rather than to importin α.3 One possibility is that VirE2 is imported via a "piggy-back" mechanism through association with VIP1 which is a nuclear protein likely having functions other than in import. It is likely that importin α in Arabidopsis is encoded by a small gene family as at least 4 members have been reported.73 Another member of this family has been found in a two-hybrid screen using a WD40 type regulatory protein as a bait.74 PRL1 when disrupted by the insertion of a T-DNA tag results in a pleiotropic phenotype conferring hypersensitivity to glucose, sucrose and hormones. PRL1 was found to interact in vivo and in vitro with ATHKAP2 which has high identity with At-IMP α, but ATHKAP2 has a truncated carboxy terminal end being about 60 amino acids shorter than AtIMP α. As with the other characterized importin αs it possesses motifs required for interaction with importin β as well as the characteristic eight conserved armadillo repeats presumably for protein-protein interactions.45

An importin α homologue (importin α1) from rice has been characterized that is 76% identical to At-IMP α. Expression of the gene in rice is suppressed by light in both etiolated seedlings and in leaves but not in roots or calli which display constitutive expression.75 Binding to NLSs was examined in vitro, and importin α1 was found to bind to the SV40 large T antigen NLS and the bipartite signal from Opaque 2. However no association was observed between importin α1 and either the yeast Matα2 signal or the Matα2-like signal from the R protein.76 This again points to some selectivity in NLS recognition in plants. Rice importin α binds to importin β from mouse, although with an affinity much lower than that of mouse importin α. Nevertheless in vitro import in permeabilized HeLa cells could be made dependent upon the presence exogenous rice importin α1 and mouse importin β indicating that rice importin α1 can function in a manner similar to that of the animal and yeast homologues.76

Importin β in plants has been less studied than importin α, although there should be a significant number of genes for importin βs by analogy with vertebrates. There are a few plant importin βs that are characterized, and the results indicate a high degree of functional conservation between kingdoms. The first importin β homologues to be reported are from rice,77 and these are designated rice importins β1 and β2. Recombinant importin β1 can interact in vitro with rice importin α1 and a second rice importin α homologue (rice importin α2). This was studied by examining mobility shifts of protein complexes on native polyacrylamide gels. Using this approach it is argued that importin β1 specifically interacts with Ran-GTP and not Ran-GDP which is consistent with the ability of Ran-GTP to dissociate the importin α/importin β import complex in animals and yeast. In permeabilized HeLa cells, exogenous rice importin α1 and importin β1 can support import in the presence of mouse Ran.78 This result plus the finding that exogenous importin β1 can bind directly to the nuclear envelope in permeabilized tobacco BY2 cells argues that importin α and β functions are conserved. It will be interesting to examine the importin βs and other components for tissue specific or light regulation as has been observed for rice importin α1 because regulation of development by light is an essential and unique feature of plants.

The Ran GTPase has been characterized in plants and homologues have been reported in Arabidopsis,79 tomato80 and tobacco81 among other species.82,83 While a direct role for Ran in nuclear import in plants has not been established, the protein is localized to the nucleus and appears to be encoded by a small gene family of at least 3 members79 that are expressed throughout the plant including tissues that are not actively growing. Furthermore, it has been demonstrated that homologues from tomato or tobacco when expressed in Schizosaccharomyces pombe can suppress the phenotype of the pim46-1 mutant which is defective in cell cycle progression.80,81 Ran is known to be involved in cell cycle control suggesting that the tomato and tobacco Ran homologues have functions analogous to those in other organisms.

Both RanGAP1 and RanBP1 can stimulate Ran GTPase activity in the cytoplasm favoring Ran-GDP in that compartment. RanBP1 has been identified functionally from Arabidopsis by using the Ran homologue AtRan1 as bait in a two-hybrid screen in yeast. Haizel et al79 found interaction with two RanBP1-like proteins (At-RanBP1a and At-RanBP1b) having 60% identity with vertebrate Ran BP1 and possessing conserved domains for Ran binding. Further study indicates that both At-RanBP1s are capable of associating in vivo in yeast with the GTP-bound form of each of the three Ran homologues from Arabidopsis (AtRan1, AtRan2, AtRan3). Neither the intracellular location nor the ability of At-RanBP1a or At-RanBP1b to stimulate GTPase activity has been reported. Likewise, neither RanGAP1 nor the nuclear exchange factor RCC1 has been characterized functionally in plants. Two putative RanGAPs have been identified from Arabidopsis, and they appear to have a unique domain not present in Ran GAPs from animals and yeast.84 The motif which has been named a WPP domain is shared with MAF1.85 MAF1 is a recently discovered protein that is composed mostly of WPP repeats and appears to be localized to the nuclear envelope via interaction with another envelope protein, MFP1.86 The WPP domain appears to be specific to plants and is hypothesized to be involved in protein-protein interaction.84 If true, RanGAP may associate with the nuclear envelope and NPCs in plants through this interaction. Table 1 describes components of the nuclear import pathway in plants that have been characterized to date.

Table 1. Components of the nuclear protein import pathway in plants.

Table 1

Components of the nuclear protein import pathway in plants.

It is increasingly apparent that there is functional conservation of the basic nuclear import pathway in plants, vertebrates and yeast. However many of the interesting biological questions will no doubt reside in the ways that plants have adapted the basic nuclear import pathways to fit their sessile photosynthetic life style. Exceptions have been noted already (importin β independence of At-IMP α, a potential alternative pathway for VirE2 import, differences in NLS selectivity) and others will be discussed below.

Systems to Study Import in Plants

The essential breakthrough that permitted the biochemical identification and purification of the factors now known to be involved in nuclear translocation of proteins and other substrates was the development of an in vitro import system utilizing permeabilized animal cells.87,88 The method relies on the fact that plasma membranes from animals can be selectively permeabilized with digitonin, a reagent that aggregates to form pores upon binding to cholesterol. This sterol is abundant in the plasma membranes of animal cells but not in other membranes such as the nuclear envelope. The effect of the reagent is to permit the selective depletion of soluble factors from cultured cells while leaving the integrity of the nuclear envelope intact. Nuclear import thus occurs only by authentic facilitated translocation rather than by simple diffusion into damaged nuclei through tears in the nuclear envelope. Import can be directly visualized by microscopy following the addition of fluorescently labeled NLS-containing proteins. Another accepted method to examine import is microinjection into Xenopus oocytes or mammalian cells which is analytical and not amenable to fractionation of components. Other approaches that have been reported include the use of purified nuclei or nuclei mixed with Xenopus cytosol extracts (for a review see ref. 4); however, the ease of the permeabilization assay resulted in broad acceptance.

Although digitonin is not the reagent of choice in yeast and plants due to differences in membrane composition, the principle of selective permeabilization has been utilized successfully. A method in yeast was developed in which the plasma membrane of cell wall-less spheroplasts was selectively permeabilized via a simple freeze-thaw technique.89 As in animal cells, import is dependent upon ATP (which can be converted to GTP for import), temperature, and the presence of exogenous cytosol (from yeast or mammalian cells). Again, alternative methods have been reported.90

In plants the development of in vitro import systems was difficult technically due to the fact that plant cells possess a thick cell wall and are highly vacuolated. The first report of in vitro import was an antibody cotranslocation assay in which antibodies to G-box binding factors (GBFs) are translocated into the nucleus (presumably by association with the GBFs) of Triton X-100 permeabilized parsley cells.91 The results indicate that GBFs involved in light-regulated gene expression are imported in response to light and that import is ATP and temperature dependent. The assay is indirect however relying of protease protection of antibody associated with the nucleus. Two groups reported the development of direct import assays using evacuolated protoplasts from tobacco.92 The approaches were similar with Merkle et al49 using Triton X-100 to permeabilize the plasma membrane, whereas Hicks et al48 used an osmotic shift to achieve permeabilization without detergents. In both cases, direct visualization of fluorescent import substrates indicates specific import that is dependent upon GTP hydrolysis and is specific for proteins containing functional NLSs. Some interesting differences are apparent between import in plants and import in animals and yeast. Import in plants is only partially inhibited on ice compared to an almost complete block in yeast and animals (perhaps an adaptation), and import was not blocked by WGA as in animals (perhaps due to the unusual NPC modifications). The most fundamental difference, however, is that import in permeabilized plant cells can occur in the absence of exogenous cytosol. This is not to suggest that cytoplasmic factors are not required in plants as in animals and yeast. However, a significant fraction of specific import factors may be tightly associated with cellular structures such as the cytoskeleton that are not disrupted by the permeabilization techniques used. There is support for this notion.

By direct observation in permeabilized protoplasts48 and by fractionation of purified nuclei45 it is clear that in the presence of high concentrations of Triton X-100 significant fractions of At-IMP α remain in the cytoplasm and in association with the nuclear envelope in addition to a soluble pool. Whereas these observations provide an opportunity to investigate potential mechanisms for retention of At-IMP α, unfortunately they limit the utility of the assays for the identification of essential import factors in plants. Alternatives have been used most of which are heterologous systems. As noted Ballas and Cytovsky (ref. 72) have used the yeast permeabilized system to demonstrate the function of AtKAPα in the import of VirD2. Other heterologous systems have been used to examine Agrobacterium Vir proteins including Xenopus, Drosophila, mammalian, and yeast cells.57–59 Other examples previously cited are the functional characterizations of At-IMP α and rice importins α and β in permeabilized HeLa cells. An alternative heterologous approach is to utilize plant cytosol extracts to support import in animal cells. This approach was examined, and it was found that cytosolic extract from petunia could in fact support import in permeabilized HeLa cells. As in animal cells import is temperature dependent, requires GTP hydrolysis and is blocked by WGA.93 The final approach that has proven useful is microinjection of import substrates into the stamen hairs of Tradescantia. This was valuable in characterizing the nuclear import of VirE2-single stranded DNA complexes.94 VirE2 facilitates Agrobacterium infection by associating with pathogen DNA in the plant cell cytoplasm and assisting in its nuclear import. Import of the complexes was found to be dependent upon GTP hydrolysis and was inhibited by WGA. The inconsistency of WGA inhibition upon injection compared to a lack of inhibition in permeabilized cells in unclear. Perhaps the large mass of the VirE2-protein complexes renders them more susceptible to import inhibition than simple protein substrates.

Regulated Protein Import in Plant Development

Plants lead a sessile life style and thus have evolved sensitive mechanisms to control development and withstand environmental challenges. A large number of gene products are induced in response to light, which is an essential developmental stimulus. Seedlings respond to light by undergoing photomorphogenesis, a process that includes altered morphology (shorter stems, leaf development), the development of chloroplasts and induction of the photosynthetic machinery (greening). Even fully differentiated plants must continually respond to quantitative and qualitative differences in light and other environmental challenges such as temperature fluctuations. For the purposes of illustrating the important roles that nuclear protein import play in such responses, we will discuss several interesting examples in which regulation of import potentiates a response to environmental cues.

Photomorphogenesis

Genetic screens have identified components in light signaling that include photoreceptors (For a review see ref. 95) and downstream components that couple light signals to gene expression during photomorphogenesis (see for example refs. 96-99). The understanding of photomorphogenesis including the mechanisms of light-modulated gene expression is major area of plant biology beyond the scope of this Chapter but recently reviewed in detail.100–103 In Arabidopsis, screens for defective or inappropriate responses to light have resulted in mutants that are photomorphogenic (ie they possess a light-grown phenotype) in complete darkness and define at least 11 loci known as the COP/DET/FUS loci.1,104,105 One protein that acts as a negative regulator of photomorphogenesis is COP1. Mutations in COP1 result in plants that develop a light-grown phenotype including chloroplast development in complete darkness. When expressed as a GUS fusion protein in Arabidopsis, COP1 localizes primarily to the nucleus in the dark in leaves and shoots but is exclusively nuclear localized in the roots.104 However in the light COP1 partitions between the nucleus and cytoplasm in leaves and shoots. COP1 possesses, in addition to a bipartite NLS, a zinc-binding domain, a coiled-coil domain, and WD-40 repeats. Recent characterization of COP1 domain structure indicates that the amino terminal region containing the zinc-binding domain is essential for basal function, whereas the carboxyl terminal domain is necessary for the repression of photomorphogenesis in the dark.105 Although the precise mechanism by which COP1 targeting is regulated by light is not fully understood, a potential route by which photomorphogenic repression is achieved is via interaction of COP1 with the basic leucine-zipper (bZIP) transcription factor Hy5. Hy5 binds to G-box containing promoters for light regulated genes such as ribulose bisphosphate carboxylase/oxygenase small subunit (RBCS) and chalcone synthase (CHS). It has been identified as a suppressor of the cop1 mutant and has been shown to interact physically with COP1 protein.106 Several studies indicate that Hy5 is an exclusively nuclear protein that is abundant in the light but degraded in the dark. Furthermore, the degradation is clearly dependent upon interaction with COP1 in the nucleus because a truncated Hy5 protein lacking a COP1-interaction domain is no longer degraded in the dark.1 Thus, COP1 functions as a repressor of photomorphogenesis by signaling the selective degradation of downstream affectors including Hy5 which participates gene expression essential for development. Recent evidence suggests that degradation of HY5 is mediated by the ubiquitin pathway by interaction with a COP1-containing proteosome107 and that HY5 degradation can be further modulated by phosphorylation of the COP1 interaction domain.108 Expression of HY5 is interestingly itself under negative regulation in the light by the action of a recently discovered calcium-binding protein, SUB1109 The story is more complicated as COP1 appears to interact via its coiled-coiled domain with yet another protein (CIP1) that is cytoplasmic and is capable of interacting with the cytoskeleton.110 One hypothesis is that CIP1 is involved in the retention of COP1 in the cytoplasm in the light essentially excluding it from the nucleus.

The perception of light involves multiple photoreceptors that detect blue light (cryptochrome), UV-B, and red light (phytochrome).95,111 The best characterized family of receptors are the phytochromes which are soluble proteins possessing a tetrapyrrole chromophore for activation by red light. After light absorption, the inactive form of phytochrome (Pr) is converted to the active far-red light absorbing form (Pfr) that participates in signal transduction and expression of genes involved in photomorphogenesis. The morphological consequences of red light perception include characteristic hypocotyl shortening and red light dependence of seed germination. There are five genes encoding phytochromes in Arabidopsis (phy A through phy E). The best characterized are phy A and phy B each of which detects red light in different ways as developmental cues. Phy A is rapidly degraded in the light and much more abundant in dark-grown plants than phy B. PhyA is responsible for the so-called very low fluence responses (VLFR) and for absorption of continuous far-red light known as the high irradiance responses (HIR). Phy B is stable in the light and is responsible for the detection of red light known as the low fluence responses (LFR) which are reversible by far-red light.95

For the induction of gene expression in response to light signals there must be communication between the soluble photoreceptors (that are for the most part cytoplasmic) and the nucleus. Recent experiments indicate that phy A and phy B are transported from the cytoplasm to the nucleus in response to red light. Rice phy A and tobacco phy B were fused to green fluorescent protein (GFP) and overexpressed in tobacco.112 When adapted to growth in the dark, phy A-GFP and phy B-GFP were not detected. However upon exposure to as little as 5 min of far-red light phy A-GFP (i.e., VLFR) localized to the nucleus. In contrast, upon exposure to red light (i.e., LFR), but not far-red light, Phy B-GFP was found in the nucleus and the localization was reversible upon irradiation by far-red light. Furthermore, the nuclear localization of phy B is dependent upon the presence of the chromophore. The kinetics of the light-dependent relocalization have been examine in detail.113,114 These results are consistent with the biological activities of phy A and phy B and indicate that their regulated targeting in response to the quality of the red light is an important step in phytochrome signaling. The mechanism of import inhibition in the dark is hypothesized to involve the masking of putative NLSs within the carboxyl terminus via structural changes that are dependent upon the presence of functional chromophore or perhaps a specific retention of Pr in the cytoplasm in the dark (for discussion see ref. 112).

The light-regulated nuclear import of several classes of transcription factors has also been described recently and may provide additional pathways for the control of photomorphogenesis. Nuclear translocation of the common plant regulatory factor (CPRF) proteins in parsley cells has been shown to be under red light control and is far-red reversible.115 The CPRFs are bZIP transcription factors that bind to G-box elements found adjacent to many light-regulated genes. Of the three CPRFs that have been examined (CPRF1, CPRF2, CPRF4), CPRF2 was found in the cytoplasm in the dark and relocated to the nucleus in the light. Immunolocalization indicates that phy A HIR and phy B LFR responses are involved implicating CPRFs in phytochrome signaling and provides regulation in addition to light-modulated nuclear import of the receptors. A deletion analysis of CPRF2 reveals two potential domains involved in cytoplasmic retention in the dark. Neither domain has homology to the COP1 retention factor CIP1110 nor to a retention domain in the bZIP factor G-box binding factor 1 (GBF1; discussed below).116 However, one retention domain of CPRF2 shares 25% identity with an a-helical retention domain from mammalian heat shock factor 2.115

Other examples of light-regulated nuclear import are known. Using the previously discussed parsley in vitro antibody cotranslocation assay, Harter et al91 have found evidence for a cytoplasmic pool of GBF-transcription factors involved in light-regulated gene expression. The GBFs are another class of bZIP transciption factors that bind to G-box elements and participate in light-regulated gene expression. Upon exposure to white light, GBFs were found in the nucleus, presumably due to light stimulated relocalization. More recently, Kircher et al117 have cloned several CPRFs from parsley and using the antibody cotranslocation assay find that parsley CPRF1, CPRF2 and GBF2 are translocated to the nucleus in response to UV light. GBFs have been examined further using three different Arabidopsis GBF genes fused to the reporter GUS and examined by transient expression in soybean protoplasts.116 About 50% of one fusion protein (GUS:GBF2) was found in the nucleus in the dark, whereas this increased to about 80% upon exposure to blue light. Deletion analysis of a different fusion protein (GUS:GBF1) resulted in an increase in nuclear localization from a maximum of 50% to about 90%. This analysis may have identified a region involved in cytoplasmic retention of GBFs in the dark. One caveat is that GUS:GBF1 itself is not under light control being about 50% nuclear under all conditions tested. Given the importance of light in plant development additional examples of modulated nuclear import will no doubt be identified.

Other Examples of Regulated Import

Plants must continually respond to environmental and pathogen challenges and examples indicating the involvement of regulated import are being discovered. In tomato, the import of several heat shock transcription factors (Hsfs) requires protein-protein interaction. The expression of HsfA1 is constitutive but is accompanied by the expression of several heat shock inducible forms called HsfA2 and HsfB1. HsfA2 has been shown in tomato and tobacco protoplasts to be mostly cytoplasmic upon heat shock, even though related factors such as HsfA1 are translocated to the nucleus under these conditions. If a short region is deleted from the carboxyl terminus HsfA2, the protein is strongly localized to the nucleus.118 Interestingly, when coexpressed with HsfA1 in tomato protoplasts, HsfA2 is efficiently translocated following heat shock. The cotranslocation is dependent upon the physical interaction of HsfA1 and HsfA2 as demonstrated by coimmunoprecipitation and a two-hybrid assay.119 The stress induction of HsfA2 and its interaction with constitutively expressed HfA1 to form a transcriptionally active heterodimer provides a mechanism for dynamic changes in the intracellular distribution of HsfA2.

We have already discussed aspects of the Agrobacterium system in which the pathogen utilizes endogenous plant import components to assist is the infection process. Nuclear import in plants can also be under viral control. An interesting example occurs in plants infected with the squash leaf curl virus (SqLCV), a geminivirus (for review see refs. 2, 120). The virus encodes two movement proteins, BR1 and BL1, which cooperativey participate in cell-to-cell spread of the virus. BR1 is an NLS-containing protein that shuttles between the nucleus and cytoplasm and binds to single-stranded DNA. BL1 is localized to peripheral regions of cytoplasm and appears to function in the movement of the BR1:viral DNA complexes across the cell wall to adjacent cells. When expressed transiently in tobacco protoplasts BR1 is strongly localized to the nucleus121,122 However when coexpressed with BL1, BR1 is relocalized to the cell periphery via specific interaction between the proteins122 providing a mechanism for delivery of viral genomes to the cell periphery for cell-to-cell spread. Other examples of viral protein nuclear import and cytoplasmic retention controlling nuclear import in viruses exist.123,124

Besides the specific examples of regulated nuclear import cited above, there is almost certainly broader control of development and environmental responses through modulation of the nuclear import apparatus itself. For example in rice, it is known that light exposure results in the down-regulation of importin α in leaves and dark-grown seedlings.75 Potentially broad control of import in plants could be imparted through phophorylation, which has been clearly implicated in both overall control of the cell cycle and in the specific regulation of imported proteins in animals and yeast (for reviews see refs. 4, 10, 125).

Recent Advances in Plant Nuclear Translocation

Several recent advances in our understanding of nuclear protein translocation in plants are worthy of mention as they have a potential impact of the field in general.

Targeting to the NPC

Many of the essential components of the import pathway have been identified animals and yeast, and are beginning to be identified in plants. In addition the NPCs have been the focus of intense investigation in animals and yeast. One fundamental question that has received little or no attention is how proteins in the cytoplasm are targeted to the NPCs for import. The notion that proteins are freely soluble in the cytoplasm where they associate with importins and diffuse to the NPCs for translocation is too simplistic. It is known that organelles and mRNAs can be transported along the cytoskeleton to specific sites.126,127 In fact, animal viruses can be targeted to the nucleus along microtubules,128 and there are strong indications that plant viral movement proteins can associate with the cytoskeleton.2,129 Could the cytoskeleton play a role in transporting complexes to the NPC for nuclear import?

Immunolocalization of At-IMP α in tobacco protoplasts is suggestive of cytoskeleton, extending from the nucleus throughout the cytoplasm to the cell periphery.45 In addition as noted previously, At-IMP α like other importin αs contains hydrophobic armadillo repeats implicated in protein-protein interactions including association with the cytoskeleton130. Furthermore, At-IMP α cannot be fully depleted from the cytoplasm of permeabilized cells indicating a tight association with intracelluar components.48 These observations prompted Smith and Raikhel7 to investigate the role of the cytoskeleton in NPC targeting using double-immunofluorescence and confocal microscopy.7,46 importin α was found to colocalize with microfilaments and microtubules in tobacco protoplasts, whereas depolymerization of cytoskeleton results in loss of the cytoskeleton-like staining pattern. Depolymerization of microtubules results in diffuse cytoplasmic staining (Figure 2). Interestingly, depolymerization of microfilaments results in accumulation of receptor in the nucleus, suggesting that microfilaments may be involved in retention of importin α in the cytoplasm.7 An examination of At-IMP α association in vitro in a cytoskeleton-binding assay indicates that association with microtubules and microfilaments requires the presence of a functional NLS. The NLS-dependent association of At-IMP α with cytoskeleton may represent a mechanism for the assembly and transport of import complexes to the NPC. Based upon the data, a working model has been proposed46 in which microfilaments serve as sites for assembly of importin α-NLS protein complexes (Figure 3). Transport to the NPC would likely require the participation of a microtubule motor protein. It is possible that proteins translated from polysomes associated with the cytoskeleton could be assembled into complexes following synthesis. The model is supported by observations of the movement of NLS-containing substrates along neurons toward the nucleus, which is microtubule dependent.131 It is unclear at this time what role importin β would play in the formation of cytoskeletal import complexes. Although At-IMP α appears to function in import in an importin β-independent manner, this is probably not true for other importin αs. Other connections between importins and the cytoskeleton are becoming apparent. For example, it is now known that importin β can inhibit microtubule assembly in Xenopus egg extracts, and it is suggested this serves to suppress aster assembly until interaction with Ran-GTP releases importin β and assemble can proceed during mitosis.132

Figure 2. Colocalization of importin α with microtubules in the cytoplasm of tobacco cells.

Figure 2

Colocalization of importin α with microtubules in the cytoplasm of tobacco cells. Fixed protoplasts were double immunolabeled for tubulin (Tubulin panels) and importin α (Importin panels) and examined for coalignment (Overlay panels). (more...)

Figure 3. Diagram depicting hypothetical model for intracellular retention and translocation of import complexes prior to nuclear import.

Figure 3

Diagram depicting hypothetical model for intracellular retention and translocation of import complexes prior to nuclear import. Actin microfilaments serve as a site for assembly of import complexes. importin α probably does not bind to microfilaments (more...)

Nuclear Export

Recently the nuclear shuttling protein BR1 from SqLCV protein has been examined for a nuclear export signal (NES) that would permit its export from the nucleus as has been found in the viral protein HIV Rev133 and others.134,135 This signal, like NLSs, is not strictly conserved, but is a leucine-rich hydrophobic sequence of 10 to 13 amino acids. Such as motif was found within BR1, and when its three leucine residues were mutated to alanines, viral pathogenicity was lost indicating the essential nature of these residues.9 It was further reasoned that only export competent BR1 protein could be relocalized to the cell periphery by association with BL1. In fact, GUS-BR1 fusion proteins when coexpressed in tobacco protoplasts are only relocalized to the cell periphery when the NES is present. Fusion proteins without the NES remain in the nucleus. Using this assay it was demonstrated that the NES from the Xenpous transcription factor IIIA can functionally replace the endogenous NES of BR1 and even restore viral infectivity. A homologue of the NES transporter CRM1 also known as exportin 1135–139 was recently cloned from Arabidopsis (AtXPO1) and characterized functionally in a yeast two-hybrid assay. The AtXPO1 interacts with the functional NES from AtRanBP1a as well as the NES from HIV Rev.140 In another protoplast expression system utilizing an NLS and NES fused to GFP, the NES from HIV Rev functions in export, which is inhibited by leptomycine B as in animal cells. The NES from AtRan BP1a, but not a version in which three leucines were mutated to alanines, also functions in this assay. These are the first examples in plants of characterized NESs and suggest that along with the protein import pathway, there is functional conservation with animals and yeast. Again, the interesting biology will reside in the details of how such pathways are utilized in plants, and the development of a straightforward assay for nuclear export in plants should encourage progress.

Conclusions

The study of protein import in plants is beginning to yield insight into not only the similarities with other kingdoms, but also the interesting differences that we have described throughout this Chapter. Plants are essential to all life on our planet and are the foundation for our food chain. Protein import processes and their role in development and environmental responses are essential to our understanding of plant biology, an important goal in itself. As our knowledge increases about nuclear protein import in plants, contributions to our general understanding of these processes in all organisms will increase.

Some of the important areas to be addressed in the future include:

  1. The complete characterization of import components from higher plants as has been under way
  2. Answers to the question, is there an importin β-independent pathway in plants?
  3. The further investigation of plant NPCs
  4. Taking full advantage of pathogens such as Agrobacterium and viruses in understanding pathogenesis and import and export pathways
  5. A focus on the regulation of import in essential developmental pathways in plants such as photomorphogensis, stress responses, and perhaps phytohormone signaling
  6. The molecular mechanism of import complex targeting to the NPCs for translocation including the development of a system to examine NLS protein movement along microtubules and a search for factors that may mediate complex association with the cytoskeleton

There is much to be learned and the future will surely present opportunities for new discovery.

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