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Plant Physiology, April 1999, Vol. 119, pp. 1157–1163, www.plantphysiol.org © 1999 American Society of Plant Physiologists
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
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 signaldependent 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 cellcycle 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, uridinerich 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 b-like proteins, in conjunction with a small GTPase, Ran/TC4 (for
reviews, see Görlich, 1997; Nigg, 1997). The importin b-like
proteins function as the nuclear signal receptors for specific
1
This work was supported by the U.S. Department of Energy
(grant no. DE-FG02-91ER20021).
* Corresponding author; e-mail [email protected]; fax
1–517–353–9168.
import and export pathways; RanGTP regulates the binding of these receptors to the cargo bearing the nucleartargeting 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 b and a is
required. In this pathway, the importin a-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
Abbreviations: NLS, nuclear localization signal; NPC, nuclear
pore complex.
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Smith and Raikhel
Plant Physiol. Vol. 119, 1999
into two distinct steps—docking 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 a
and b binds NLS-containing proteins in the cytoplasm via
the NLS-binding region of importin a, 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 b-subunit. In yeast
and vertebrates, importin a requires importin b for highaffinity interaction with NLSs. Also, importin b can function alone as an import receptor for a subset of NLS
proteins.
Translocation of the trimeric complex through the NPC
requires free GTP and a small GTPase, Ran, in the GDPbound 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 b terminates import by
releasing the importin a/NLS-protein complex into the
nucleoplasm. Subsequently, importin a dissociates by an
unknown mechanism from the NLS-containing protein,
and the importin a- and b-subunits are exported to the
cytoplasm, where they can participate in another cycle of
import.
Importin b is probably exported to the cytoplasm in a
complex with RanGTP. This complex is dissociated in the
cytoplasm through the action of importin a and a set of
RanGTP-activation proteins, RanGap1 and RanBP1, which
are found exclusively in the cytosol. Export of importin a is
facilitated by a heterodimer consisting of importin b, called
Cas, and RanGTP. Cas binds to importin a 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 a in the absence of
RanGTP, which allows the formation of the importin a/b
heterodimer in the cytoplasm. Thus, the NLS protein import cycle can continue.
Figure 1. A schematic representation of NLS protein import. The NLS
docking step of import is mediated by the importin a/b heterodimer.
The a-subunit binds to NLSs and the b-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 b and terminates import by
releasing the a/NLS-containing protein into the nucleoplasm. After
importin a dissociates from the NLS-containing protein, it is exported
by the Cas/RanGTP complex. Importin b is probably exported with
RanGTP. Dissociation of these complexes occurs by the RanGTPactivating proteins RanGap1 and RanBP1, which are found only in
the cytoplasm. This allows the formation of the importin a/b complex, and the cycle of NLS protein import 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-100permeabilized 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).
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Targeting to the Nucleus
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 a homolog (At-IMPa) show that importin a 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 a 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 pe-
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riphery 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-Mr 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-Mr 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-Mr 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 a
and importin b. Homologs of importin a have been identified in plants. Immunolocalization of importin a 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 a homolog, At-IMPa, 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-IMPa may represent a
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Smith and Raikhel
unique class of NLS receptors that have not been identified
in vertebrates and yeast. Unlike yeast and vertebrate importin a-subunits, At-IMPa recognizes NLSs with high
affinity, and, even more intriguing, At-IMPa can facilitate
NLS protein import in the absence of a b-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 a-subunits.
Another Arabidopsis importin a protein, At-KAPa,
which is 94% identical to At-IMPa, was recently found to
complement a temperature-sensitive mutation in the yeast
importin a protein SRP1, suggesting that plants and yeast
share conserved NLS protein import pathways (Ballas and
Citovsky, 1997). Recently, four more importin a-like proteins were identified in Arabidopsis (Schledz et al., 1998),
suggesting that importin a is probably encoded by a small
gene family in higher plants.
Unlike At-IMPa, the rice importin a-subunit recognizes
only mono and bipartite NLSs. In addition, this rice
a-subunit stimulates NLS protein import in conjunction
with a mouse importin b-subunit in the vertebrate import
system. This observation suggests that a conserved NLS
protein import pathway mediated by the a/b heterodimer
also exists in plants (Jiang et al., 1998). More importantly,
characterization of At-IMPa suggests that plants possess an
importin b-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
Plant Physiol. Vol. 119, 1999
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 AtRanBP1b. 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 a 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 a 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.
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Targeting to the Nucleus
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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 a 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 lectinaffinity 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.
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 a associates
with the cytoskeleton in plants. Immunolocalization of AtIMPa 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-IMPa 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 a contains highly hydrophobic armadillo repeats
of 42 amino acids in length; these are implicated in proteinprotein interaction (Hicks et al., 1996). Thus, importin a
IMPORTIN a INTERACTS WITH THE
CYTOSKELETON IN PLANTS
Figure 2. Importin a colocalizes with microtubules in the cytoplasm
of tobacco protoplasts. Most of the cytoplasmic importin a (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 a. Superimposing these two images
(c) demonstrates coalignment, shown in yellow/orange.
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
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Smith and Raikhel
could interact with the cytoskeleton, because other proteins
containing armadillo repeats have such interactions with
these structures (Barth et al., 1997).
We recently demonstrated that importin a 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-IMPa in the cytoplasm. It is interesting that
when the microtubules are depolymerized importin a
staining in the cytoplasm is diffuse. However, depolymerization of microfilaments causes At-IMPa 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-IMPa associates with microtubules and microfilaments
in an NLS-dependent manner. This association could be
essential for the transport of importin a/NLS-containing
protein complexes in the cytoplasm. The NLS-dependent
association of importin a 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 a with NLScontaining proteins. The yeast importin a-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 a in the cytoplasm. This retention mechanism may perform as an assembler of the importin
a/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 a, forming
transport complexes that are then loaded onto microtubule
tracks for transport to the NPC.
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 a does not share similarity
to known motor proteins such as kinesin or dynein.
Plant Physiol. Vol. 119, 1999
Figure 3. A schematic model for intracellular transport of the
importin/NLS-containing protein complex. In this hypothetical
model, importin a forms transport-competent complexes on microfilaments where nuclear proteins are synthesized. Protein “X,” which
anchors importin a to the microfilaments, could be Act2-like protein,
which interacts with importin a. 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.
Future work should be directed toward understanding
mechanistic details of import in plants. For example, is the
importin b-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
a/NLS protein complexes in the cytoplasm. An NLSprotein transport system should be developed to characterize the movement of NLS-containing proteins along microtubules. The identification of cytoskeleton-binding
factors that mediate importin a 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.
ACKNOWLEDGMENT
We thank Dr. Glenn Hicks for critical reading of the manuscript.
Received December 9, 1998; accepted January 21, 1999.
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