1 Department of Anatomy and Cell Biology, University of Florida, College of
Medicine, Gainesville, FL 32610, USA
2 MRC Laboratory of Molecular Biology, Hills Rd, Cambridge CB2 2QH, UK
* Author for correspondence (e-mail: feldherr{at}anatomy.med.ufl.edu )
Accepted 1 May 2002
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Summary |
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Key words: Nuclear transport, Nuclear pore complex, Amoebae
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Introduction |
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There are considerable data demonstrating that the basic transport process
outlined above functions in organisms as diverse as yeast and vertebrates.
However, specific components of the transport machinery exhibit different
levels of conservation. The transport receptors that function in the import
and export of proteins through the nuclear pores are all members of the
importin-ß (or karyopherin-ß) superfamily
(Wozniak et al., 1998;
Gorlich and Kutay, 1999
;
Jans et al., 2000
). The
characteristic features of these transport receptors are an N-terminal
RanGTP-binding domain, a relatively large mass (approximately 90-140 kDa), and
an acidic isoelectric point. In addition, the receptors contain binding sites
for specific NLSs, NESs or adaptors, and are able to interact with
nucleoporins, mainly by binding to FG motifs (see below). Fourteen and 21
importin-ß family members have been identified in yeast and humans,
respectively. Of the yeast members, five are involved in export, and the
remainder function in protein import. Kutay et al. suggested that the greater
number of receptors in vertebrate cells could (1) increase redundancy in the
pathways available for specific signals or classes of signals or (2) provide
pathways for transport substrates that are not present in yeast
(Kutay et al., 2000
).
Significant differences have also been detected in the organization and
composition of the nuclear pore complexes. In yeast, the pore complex has a
mass of about 66 MDa (Yang et al.,
1998) and contains multiple copies of approximately 30 different
nucleoporins (Rout et al.,
2000
). The pore complex in vertebrate cells has a mass estimated
to be 125 MDa, and it is thought to be made up of over 50 different proteins.
A detailed comparison of the nucleoporins in the two organisms has recently
been published by Vasu and Forbes (Vasu
and Forbes, 2001
). Six of the known vertebrate nucleoporins show
strong homology with yeast nucleoporins; however, the majority are only weakly
homologous or distantly related. Despite this diversity, one feature that is
common to both yeast and vertebrate nucleoporins are FG-repeat motifs. This
general classification includes FG, FxFG or GLFG repeats.
There are data showing that FG-containing nucleoporins are present
throughout the pore complex (e.g. Grote et
al., 1995; Stoffler et al.,
1999
; Rout et al.,
2000
; Stewart et al.,
2001
); some are distributed symmetrically, whereas others are
localized specifically on the nuclear or cytoplasmic face of the complex.
Consistent with this distribution, there is evidence that the FG repeats are
required for translocation through the pores. For example, binding studies,
reviewed by Ryan and Wente, have revealed that all of the transport receptors
that have been studied interact with one or more FG-containing nucleoporins
(Ryan and Wente, 2000
).
Although a number of receptors bound to the same nucleoporins, differences in
the overall binding patterns have been detected. Microinjection or
overexpression of peptides containing FG-repeat domains interferes with
nuclear transport, providing further evidence that FG motifs are involved in
the translocation (Bastos et al.,
1996
; Iovine et al.,
1996
; Stutz et al.,
1996
).
Currently there are three general models for translocation through the
pores, all of which are centered around interactions between FG repeats and
transport receptors. Rout et al. have proposed that pore-associated
FG-containing filamentous proteins, assisted by Brownian movement, exclude
substances that lack FG-binding domains, but, at the same time, bind to
receptor-cargo complexes for subsequent diffusion through the pores
(Rout et al., 2000). Ribbeck
and Gorlich suggested that the transport channel located in the center of the
pore complex is occupied by a meshwork of FG repeat nucleoporins that
functions as a semi-liquid phase into which receptor-cargo complexes,
containing FG-binding sites, could partition and thereby translocate through
the pores (Ribbeck and Gorlich,
2001
). A third model (reviewed by
Stewart et al., 2001
) proposed
that receptor-cargo complexes initially concentrate at the cytoplasmic face of
the pores by binding to FG repeats. Transport across the envelope would then
involve sequential steps of adsorption and desorption to FG motifs that line
the central channel of the pores. It is also possible that directional
migration across the envelope is enhanced by an `affinity gradient' for
different nucleoporins within the pore complexes
(Ben-Efraim and Gerace, 2001
).
Distinguishing among these models remains a major challenge in the field.
It is likely that the major evolutionary changes in nuclear transport capacity needed to accommodate the additional regulatory requirements of higher organisms are caused by increases in the number of transport receptors and the complexity of the pore complex. The translocation process itself appears to be more highly conserved. Thus, transport in both yeast and vertebrates is typically Ran dependent, consistent with the conserved Ran-binding domain being located in the N-terminus of the receptors. FG-containing nucleoporins, which are also required for translocation, are abundant in yeast and vertebrate cells. These similarities raise the possibility that there is an underlying, universal pathway for translocation through the pores.
This study is a continuation of an earlier investigation on nuclear
transport in Amoeba proteus. The basic premise of these experiments
is that evolutionary changes in the properties of the transport machinery are
related to the changing regulatory requirements of cells as complexity
increases. Thus, an understanding of the nucleocytopasmic exchange in
primitive systems should help to distinguish between the basic, essential
elements of the transport process and regulatory elements that evolved to
satisfy the requirements of more complex organisms. Previously
(Feldherr and Akin, 1999), it
was determined that the nuclear transport apparatus in amoebae is different
from vertebrate cells. First, the functional diameter of the central transport
channel is smaller in amoebae. This was established by analyzing the import of
different size gold particles that were coated with BSA conjugated to peptides
containing the classical large T NLS. In amoebae, particles larger than 140
Å in diameter were essentially excluded from the nucleoplasm, whereas in
vertebrates the exclusion limit is approximately 230 Å. Presumably, this
reflects differences in the nature and organization of the nucleoporins. It
was suggested (Feldherr and Akin,
1999
) that variations in functional pore size could be related to
differences in the dimensions of the ribosomal subunits that exit the nucleus.
Second, although large T NLSs facilitated the transport of gold through the
pores in amoebae, particles coated with conjugates containing bipartite NLSs
were largely excluded from the nucleus. In vertebrate cells both signals
utilize the same receptor (the importin
/ß heterodimer) and are
equally effective in initiating transport. Data were also obtained suggesting
that the large T NLS mediates nuclear export as well as import. Thus, there
also seem to be differences in the specificity and activity of the transport
receptors.
The present report compares the molecular mechanism of protein import in A. proteus with that of vertebrate cells. It was initially found that FG repeats are present in amoebae nucleoporins and that these repeats, along with Ran, are necessary for signal-mediated nuclear import. Although proteins containing the classical large T NLS were efficiently transported into the nucleoplasm in amoebae, substrates bearing other common vertebrate import signals, specifically the M9 shuttling sequence and the bipartite NLS, were retained in the cytoplasm unless the corresponding vertebrate transport receptors were also present. These results demonstrate that the nuclear protein import machinery in A. proteus is able to recognize and utilize vertebrate receptors, which argues in favor of a highly conserved translocation process.
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Materials and Methods |
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Western blot analysis of Ran
100 µl of packed amoebae were collected by low speed centrifugation in
amoebae medium to which was added 0.1 mg/ml each of leupeptin, aprotinin and
pepstatin. An equal volume of 2x sample buffer [125 mM Tris (pH 6.8),
20% glycerol, 10% BME, 4.6% SDS, and 0.02 mg/ml bromophenol blue] was added to
the pellet, and the cells were homogenized and boiled for 5 minutes. Samples
were then run on a 10% SDS-polyacrylamide gel
(Laemmli, 1970). A431 cell
lysate (Transduction Labs, Lexington, KY) served as a positive control for the
Ran antibody.
The gel was blotted onto nitrocellulose (MSI, Inc., Westborough, MA) for 1
hour at 12 amps. The blot was incubated in blocking buffer (1% BSA, 10 mM Tris
(pH 7.5), 100 mM NaCl, and 0.1% Tween 20) for 12 hours at 4°C. This was
followed by incubation for 1 hour at room temperature in 1:5000 dilution of
monoclonal antibody against human Ran (Transduction Labs) in blocking buffer.
The blot was then rinsed for 1 hour and stained using
alkaline-phosphatase-conjugated rabbit anti-mouse IgG (Sigma, St. Louis, MO),
according to Blake et al. (Blake et al.,
1984).
Transport factors
Previously described methods were used for the bacterial expression and
purification of human importins and ß
(Bayliss et al., 2000b
),
His-tagged transportin (Pollard et al.,
1996
), canine Ran (Stewart et
al., 1998a
), rat NTF2 and its W7A mutant
(Bayliss et al., 1999
), the 18
FxFG repeat construct of yeast nucleoporin Nsp1p
(Clarkson et al., 1997
) and the
11 FxFG repeat region of rat nucleoporin p62
(Buss and Stewart, 1995
). All
proteins were over 95% pure by SDS-PAGE stained with Coomassie Brilliant Blue.
The Ran mutant Q69L was kindly supplied by Mary S. Moore, and the FxFG repeat
region of Nup153 (amino acids 618-828 of the Xenopus Nup153 sequence)
was provided by Douglass Forbes. The monoclonal antibody, MAb414, against
FG-containing nucleoporins, was purchased from Berkeley Antibody Company
(Richmond, CA).
Transport substrates
The substrates used to assay nuclear import included (1) gold particles
coated with BSA conjugated to peptides containing classical NLSs, and (2) a
recombinant protein constructed by fusing GST, GFP and the M9 transport
signal.
Signal peptides were synthesized by the University of Florida protein core
facility. The peptides, CGGGPKKKRKVGG and CGGG-
AVKRPAATKKAGQAKKKKLNGG, contain, respectively, the SV40 large T NLS
(Kalderon et al., 1984) and
the bipartite nucleoplasmin NLS (Robbins
et al., 1991
). The signal sequences are underlined. Conjugation of
the peptides with BSA (Sigma, St Louis, MO) was performed as described
previously (Lanford et al.,
1986
). It was estimated, using SDS-PAGE analysis, that an average
of eight peptides were cross-linked to each BSA molecule.
Two GST/GFP fusion proteins containing either the wild-type (wt) or mutant
(mt) M9 nuclear transport sequence, designated GST/GFP/M9wt and GST/GFP/M9mt,
respectively, were prepared by the University of Florida Molecular Services
Core Laboratory. The M9 domain is a bifunctional transport signal that is
required for the nuclear import and export of the heterogeneous nuclear
ribonucleoprotein A1 [hnRNP A1; (Michael
et al., 1995)]. The amino acid sequence of the M9 signal is
NNQSSN(FGPM)KGGNFG-GRSSGPYGGGGQYFAKPRNQGGYG
(Siomi and Dreyfuss, 1995
). In
the M9 mutant peptide, the four amino acids in parentheses were replaced with
`AAAA' (Bogerd et al., 1999
).
The dsDNA fragment encoding the M9 peptide was generated by PCR from
overlapping primers. GST fusion vector pGEX-4T-2 (Amersham Pharmacia Biotech,
Inc., Piscataway, NJ) was used to make the constructs. GFP/M9(wt or mt) was
cloned into the vector's BamHI-EcoRI site, just downstream
of GST. The fusion proteins were overexpressed in F' E. coli
and purified on a glutathione-agarose column (Sigma, St Louis, MO). Purity of
the proteins was verified by SDS-PAGE.
Colloidal gold
Colloidal gold fractions, containing particles ranging in diameter from 20
to 50 Å (small gold) or 20 to 120 Å (intermediate gold) were
prepared as outlined previously (Feldherr,
1965). The gold particles were coated with either BSA-NLS peptide
conjugates (for transport assays) or transport factors (for pore localization
studies) according to a procedure reported earlier
(Dworetzky et al., 1988
). It
is estimated that the protein coat increases overall particle diameter by
about 30 Å. The coated gold preparations were concentrated approximately
200-fold using Ultrafree concentrators (Amicon Inc., Beverly, MA) and dialyzed
against amoebae medium.
Microinjection
The amoebae were microinjected using an inverted microscope and a hydraulic
micromanipulator (Narishige USA, Inc., Greenvale, NY). The cells were
immobilized in an oil chamber and injected with micropipettes that had 1-2
µm tip diameters. The amount injected was approximately 5-10% of the cell
volume.
EM and fluorescent analysis
For EM analysis, the cells were fixed for 30 minutes in 4% glutaraldehyde,
postfixed for 30 minutes in 2% OsO4, dehydrated, embedded in
Spurr's resin and subsequently examined using a JEOL 100CX electron
microscope.
Fluorescent analysis was performed using a Hamamatsu CCD camera and a MetaMorph imaging system (Universal Imaging Corporation, West Chester, PA). Images were collected with a Zeiss Planapo 25x objective lens at the time points indicated. To minimize UV damage to the cells, each exposure was less than 1 second; in addition, a number 16 neutral density filter was used.
The nuclear/cytoplasmic (N/C) gold and fluorescent ratios were obtained by analyzing equal and adjacent areas of nucleoplasm and cytoplasm.
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Results |
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Small gold particles coated with wild-type NTF2, W7A-NTF2, which contains a
mutation in the FG-repeat-binding domain
(Bayliss et al., 1999) or
antibody MAb414 against FG-containing nucleoporins, were used to assay for
FxFG moieties in the pore complex. NTF2 is a 14.4 kDa transport factor
(Moore and Blobel, 1994
;
Paschal and Gerace, 1995
) that
acts as a receptor for the nuclear import of RanGDP
(Smith et al., 1998
;
Ribbeck et al., 1998
). To
perform this function, NTF2 has binding sites for both RanGDP and FxFG
repeats. It was previously shown in Xenopus oocytes that, following
microinjection, small gold particles coated with wild-type NTF2 bound
extensively to the pore complex (Bayliss et
al., 1999
). Particles were observed along both the nuclear and
cytoplasmic faces of the complex and also within the central transport
channel. However, accumulation within the pores was significantly reduced if
the particles are coated with W7A-NTF2. In this study, similar experiments
were performed on amoebae. The cells were microinjected with the gold
preparations, incubated for 30 minutes in culture medium, and then fixed for
EM examination. As was observed in oocytes, wild-type NTF2-gold was
distributed throughout the pores (Fig.
1A). When the gold was coated with W7A-NTF2 there was a
significant decrease in the number of pore-associated particles
(Fig. 1B). Correcting for
differences in the amount of gold available in the cytoplasm, it was estimated
that the decrease in binding was approximately 60%. These results indicate
that wild-type NTF2-gold binding to the pores was caused by the presence of
FxFG repeats. We also employed the monoclonal antibody MAb414
(Davis and Blobel, 1986
) that
binds specifically to FxFG nucleoporins. The localization of MAb414-coated
gold particles to the pore complexes following microinjection
(Fig. 2) provided additional
evidence for the presence of FG-nucleoporins in A. proteus.
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Several laboratories have reported that nuclear transport is inhibited by
peptides that contain FxFG repeats. Thus, peptides containing Nup153 FxFG
moieties block importin /ß-mediated protein import
(Shah and Forbes, 1998
) and
poly (A)+ RNA export (Bastos et
al., 1996
). NTF2-dependent nuclear uptake of RanGDP is inhibited
by the FxFG-repeat region of the yeast nucleoporin Nsp1p
(Bayliss et al., 1999
).
Presumably, inhibition of transport is caused by competition between the FxFG
peptides and the nucleoporins for receptor binding sites. To determine if FxFG
repeat peptides also decrease transport in amoebae, their effect on the
nuclear import of intermediate gold coated with BSA-large T NLS conjugates was
assayed. In these experiments, FxFG-repeat regions from Nsp1p, p62 or Nup153
were injected along with the gold-large T NLS transport substrate. The
concentrations of the injected FxFG peptides were 0.5, 1.25 and 1 mg/ml,
respectively. It is apparent from Table
1, which shows the N/C gold ratios in amoebae fixed 30 minutes
after injection, that all of the FxFG peptides inhibited import
(P<0.0001 in each experiment). In parallel experiments, it was
found that all of the above FxFG peptides also inhibited import of the gold
tracer in Xenopus oocytes (data not shown). It is interesting that
the p62 peptide blocked transport, since in vitro assays failed to detect
binding of importin ß to the N-terminus (FxFG-containing domain) of p62
(Percipalle et al., 1997
).
This suggests that binding between the p62 repeat peptide and the receptor,
although unstable under the stringent in vitro conditions, is sufficient to
interfere with transport in vivo. Taken together, the NTF2-gold binding
results, and the inhibitory effects of the FxFG peptides, support the view
that FxFG moieties are present in amoebae nuclear pore complexes and function
in translocation.
|
Western blotting procedures were employed to determine if Ran is also
present in amoebae. Blots of amoebae extracts and A431 lysate, which served as
the standard, were treated with a monoclonal antibody against human Ran and
subsequently stained. Bands in the 25 kDa range were detected in both cell
types; however, the amoebae Ran appeared to have a slightly higher molecular
weight. The results are shown in Fig.
3. The Q69L mutant was then used to investigate the activity of
Ran in the translocation process in amoebae. References to similar studies in
other experimental systems can be found in Stewart et al.
(Stewart et al., 1998b). This
mutant is not stimulated by RanGAP, and, therefore, is unable to hydrolyze
GTP. Since the mutant can undergo nucleotide exchange, it exists in its
GTP-bound form when introduced into vertebrate cells, and, thus, inhibits
import of proteins containing classical NLSs by preventing the formation of
receptor-substrate complexes. In the experiments performed on amoebae, 3 mM
wild-type Ran or RanQ69L was microinjected along with transport substrate
(intermediate gold coated with BSA conjugated with large T NLS), and the cells
were fixed after 1 hour. The results are shown in
Table 2. As expected, the N/C
gold ratios obtained for substrate alone versus substrate plus wild-type Ran
were not significantly different (P=0.11); however, the Q69L mutant
caused a highly significant decrease in import (P<0.0001),
demonstrating that Ran is involved in the nuclear import in amoebae.
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Vertebrate receptors are functional in amoebae
In order to analyze the activity of vertebrate transport receptors in
amoebae, it was first necessary to identify vertebrate NLSs that (1) were
inactive or marginally active in amoebae and (2) had well characterized
receptors. With these objectives in mind, fusion proteins were constructed
that contained GST, GFP and either the wild-type or mutant M9 shuttling signal
that is present in hnRNP A1. In initial experiments using BALB/c 3T3 cells, it
was found that the GST/GFP/M9wt accumulated in the nucleus, as determined by
fluorescent analysis, within 10 minutes of microinjection, whereas
GST/GFP/M9mt remained cytoplasmic (data not shown). When tested in amoebae,
neither of the constructs entered the nucleus. However, when 2 mg/ml
transportin was injected simultaneously, the construct containing the wild
type, but not the mutant M9 sequence rapidly accumulated in the nucleus.
Fluorescent micrographs taken 60 minutes after injection are shown in
Fig. 4A. The N/C fluorescent
ratios, at the time points indicated, are plotted in
Fig. 4B. The increased nuclear
import of GST/GFP/M9wt in the presence of transportin is highly significant
(P<0.0001). Also included in
Fig. 4B is a curve showing the
N/C ratios for fluorescein-labeled BSA. Since this tracer lacks an NLS, it is
essentially excluded from the nucleus; thus, the N/C ratios are a measure of
background fluorescence from adjacent cytoplasm. The intracellular
distribution of transportin-coated small gold particles was also investigated.
Consistent with the role of transportin in import, the gold particles
accumulated within the pore complexes (Fig.
5) and were able to enter the nucleus (the N/C gold ratio after 30
minutes was 0.41).
|
|
Although the large T NLS effectively mediates nuclear import in amoebae,
the bipartite nucleoplasmin NLS, a classical import signal that also utilizes
the importin /ß pathway, is only marginally functional in these
cells (Feldherr and Akin,
1999
). Small substrates containing the bipartite NLS (fluorescent
conjugates) were able to enter the nucleus, but large substrates (gold coated
with BSA-bipartite NLS conjugates) were essentially excluded from the
nucleoplasm. To determine if vertebrate receptors could enhance the activity
of the bipartite NLS in amoebae, small gold particles coated with
BSA-bipartite NLS conjugates were injected either alone or along with importin
ß plus importin
(1 mg/ml each). As a control, BSA-coated small
gold particles that lacked an NLS were simultaneously injected with importin
/ß. The cells were fixed 60 minutes after injection. TEMs are
shown in Fig. 6, and the N/C
gold ratios are listed in Table
3. The addition of importin
/ß not only resulted in
extensive binding of the transport substrate to the pores but also caused a
highly significant (P<0.0001) increase in nuclear uptake. Importin
/ß did not facilitate the import of the control, BSA-gold
particles. Injection of importin
or importin ß alone did not
affect nuclear import of the bipartite NLS gold (data not shown).
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Discussion |
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According to available structural and molecular data, the nuclear pore
complex contains a central channel lined by nucleoporins, many of which
contain FG-repeat motifs. This channel appears to be a universal conduit used
by members of the importin ß superfamily to exchange between the nucleus
and cytoplasm. Several lines of evidence indicate that many of the FG
nucleoporins located within and also adjacent to the central channel serve as
common binding sites that function in the translocation of all receptors. For
example, in vitro studies have shown that many of the receptors bind to common
FG nucleoporins (Ryan and Wente,
2000). Kutay et al. constructed importin ß fragments that
bound to the nuclear pore complex but were not released by Ran
(Kutay et al., 1997
).
Consistent with the existence of common binding sites, it was found that these
fragments had a general inhibitory effect on nuclear transport. They blocked
the export of mRNA, U snRNA, and proteins containing the Rev nuclear export
signal, as well as the import of an M9 fusion protein. Damelin and Silver used
fluorescence resonance energy transfer to investigate the interactions between
13 nucleoporins and two different members of the importin ß family, one
importin (Kap121) and one exportin (Msn5)
(Damelin and Silver, 2000
).
They found that during translocation, the receptors bound to a subset of seven
common nucleoporins, as well as one or two specific nucleoporins. These data
suggest that in addition to universal binding sites, different receptor
pathways might also require one or more specific nucleoporins. In this regard,
Walther et al. obtained evidence that the nucleoporin Nup153 is specifically
involved in the translocation of substrates mediated by importin
/ß (Walther et al.,
2001
). Using reconstituted Xenopus nuclei, these
investigators determined that the assembly of the envelopes in the absence of
Nup153 inhibited the transport of substrates that require importin
/ß but had no effect on transportin-dependent nuclear import. Shah
and Forbes previously identified a Nup153 FG-repeat domain that bound to
importin ß and blocked the transport of classical NLS substrates but not
M9-containing substrates (Shah and Forbes,
1998
). They also identified a transportin-binding domain in
Nup153, but its general role in transport is controversial
(Nakielny et al., 1999
).
Since the importin /ß heterodimer and transportin are
functional in both vertebrate cells and amoebae, and since the pore complexes
in these organisms are similar in their basic morphology and composition, it
is likely that the translocation mechanisms are also comparable. Thus, it is
probable that the FG nucleoporins associated with the central transport
channels have equivalent functional roles, based on their potential to form
multiple weak interactions with the transport factors, thereby facilitating
their translocation through the pores. In addition, specific nucleoporins,
which are required for different receptor pathways, might also be present.
These could include nucleoporins associated with either the initial or
terminal phases of the translocation process. Alternatively, the translocation
pathway in amoebae, although fundamentally similar in design to that in higher
organisms, might be more rudimentary and contain only common FG-repeat-binding
sites. Differences could be caused by variations in the proportion of the
different FG motifs, as well as their distribution within the pore complexes.
According to this model, specific nucleoporins would then function primarily
in higher organisms, either as redundant pathways to regulate relative rates
of exchange of different receptors or as essential transport factors for
particular receptors. Additional experiments, similar to those reported here,
but using different transport receptors, should help distinguish between the
latter possibilities.
In addition to providing data regarding the evolution of the nuclear
transport apparatus, amoebae might also prove to be a useful in vivo
experimental system for studying the molecular mechanisms of nuclear
trafficking. Nuclear transport assays based on permeabilized cultured
vertebrate cells or the yeast Sacchromyces cerevesiae have allowed
the components of the transport machinery to be identified and the
interactions among these components to be explored (e.g.
Gorlich and Kutay, 1999;
Nakielny and Dreyfuss, 1999
;
Bayliss et al., 2000b
;
Wente, 2000
;
Rout and Aitchison, 2001
).
However, these experimental approaches have certain limitations. Although the
yeast system makes powerful use of molecular genetics and also allows
transport to be studied in vivo, kinetic studies, such as those involving
microinjection, are not feasible. The permeabilized cell system is useful for
investigating the effects of soluble transport factors, but they do not fully
replicate in vivo conditions. As we have shown here, in vivo transport studies
can readily be carried out in A. proteus. Moreover, the fact that the
amoebae transport machinery is unable to recognize certain vertebrate NLSs can
be exploited to analyze translocation of receptors that do not normally
function in these organisms, thereby avoiding `background' import that would
complicate interpretation of the results. This would involve the construction
of mutants that would block interactions between the receptor and specific
components of the transport machinery and subsequent analysis of the transport
of signals specific for the receptor. For example, using this approach it
should be possible to establish the importance of different nucleoporin
binding sites for transport and also determine if there is a universal
requirement for Ran, an issue that is still somewhat controversial.
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Acknowledgments |
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