2043
Journal of Cell Science 112, 2043-2048 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0431
Signal-mediated nuclear transport in the amoeba
Carl M. Feldherr* and Debra Akin
Dept of Anatomy and Cell Biology, University of Florida, College of Medicine, Gainesville, FL 32610, USA
*Author for correspondence (e-mail: [email protected])
Accepted 20 April; published on WWW 26 May 1999
SUMMARY
The evolutionary changes that occur in signal-mediated
nuclear transport would be expected to reflect an
increasing need to regulate nucleocytoplasmic exchanges
as the complexity of organisms increases. This could
involve changes in both the composition and structure of
the pore complex, as well as the cytosolic factors that
mediate transport. In this regard, we investigated the
transport process in amoebae (Amoeba proteus and Chaos
carolinensis), primitive cells that would be expected to
have less stringent regulatory requirements than more
complex organisms. Colloidal gold particles, coated with
bovine serum albumin (BSA) conjugated with simple
INTRODUCTION
The signal-mediated import of macromolecules into the
nucleoplasm in vertebrate cells occurs through the nuclear pore
complex, a supramolecular structure that has a molecular mass
of approximately 125 MDa, and is made up of 50-100 different
proteins, the nucleoporins. Many, but not all of the nucleoporins
contain FG repeat motifs, and/or O-linked GlcNAc moieties.
Import is a multistep process that is initiated by a nuclear
localization signal (NLS). The most thoroughly studied signals
are the simple NLS, a single short basic amino acid domain,
and the bipartite NLS, two basic regions separated by an amino
acid spacer. These NLSs were first identified in SV40 large T
antigen (Kalderon and Smith, 1984) and nucleoplasmin
(Robbins et al., 1991), respectively, and are commonly found
in nuclear proteins (Richter and Standiford, 1992). Both signals
utilize the same nuclear import pathway. They initially bind to
the cytoplasmic adapter importin α (also referred to as
karyopherin α), and importin β (or karyopherin β), the receptor
responsible for docking the transport complex to the
cytoplasmic face of the pores (Gorlich, 1998). There are also
alternative pathways for nuclear import; for example, RNP
proteins (Siomi et al., 1997) and ribosomal proteins (Rout et al.,
1997; Jakel and Gorlich, 1998) not only contain different NLSs,
but utilize different receptors for docking. The existence of
different pathways could allow the transport of specific
functional classes of proteins to be controlled independently.
Following the docking step, translocation occurs through a
gated, central transporter element, which allows the passage of
particles up to 240 Å in diameter. By varying the degree of
(large T) nuclear localization signals (NLSs), bipartite
(nucleoplasmin) NLSs or mutant NLSs, were used to assay
nuclear import. It was found that in amoebae (1) the
diameter of the particles that are able to enter the
nucleoplasm is significantly less than in vertebrate cells, (2)
the simple NLS is more effective in mediating nuclear
import than the bipartite NLS, and (3) the nucleoporins
do not appear to be glycosylated. Evidence was also
obtained suggesting that, in amoebae, the simple NLS can
mediate nuclear export.
Key words: Nuclear transport, Pore complex, Amoeba
dilation of the central transport channel, the size of particles
entering and leaving the nucleus can be controlled (Feldherr
and Akin, 1995b). This could be especially important in
regulating the exchange of large RNP particles (Feldherr and
Akin, 1991). The translocation step involves at least two
factors: Ran, a 24 kDa GTPase, and p10/NTF2, a 14 kDa
polypeptide (Gorlich, 1997; Moore, 1998). Ran, which is
mainly in the GDP form in the cytoplasm and the GTP form
in the nucleoplasm, provides directionality to the transport
process. There is evidence that p10/NTF2 mediates the nuclear
uptake of Ran (Ribbeck et al., 1998), and might also regulate
dilation of the central transport channel (Feldherr et al., 1998).
In addition to studies on vertebrate cells, there is a
considerable amount of data regarding the transport apparatus
in yeast. The nuclear pore complex in these cells has an
estimated mass of 66 MDa (Yang et al., 1998), approximately
half that of vertebrate cells. Technically, it has not yet been
possible to measure the functional size of the pores in yeast,
although morphological studies indicate the presence of a
central transporter that has a diameter of approximately 350
Å, which is comparable to that in vertebrate cells (Yang et
al., 1998). The yeast nucleoporins also have FG repeat motifs,
but they are not glycosylated, and show limited homology
with vertebrate nucleoporins (Fabre and Hurt, 1997). The
NLS receptors in yeast and vertebrate cells show a greater
degree of homology and, in addition, yeast contain homologs
of Ran and p10/NTF2 (Corbett and Silver, 1997). The
similarities in signal receptors and translocation factors
suggest that the transport pathways are highly conserved. The
differences in the pore complexes, on the other hand, could
2044 C. M. Feldherr and D. Akin
mean that vertebrate cells have additional regulatory
requirements that necessitate modifications in organization
and composition of these structures.
In this study, signal-mediated nuclear transport was
investigated in amoebae. Assuming that the complexity of the
nuclear transport machinery is related to the level of regulation
required for cell function, it would be expected that amoebae
would have a more primitive transport system than that found
in higher organisms. If this assumption is correct, comparative
studies should not only provide a better understanding of the
evolution of the nucleocytoplasmic exchange process, but
might also provide clues regarding the functional role of the
different elements of the transport apparatus.
Previous studies relating to nuclear transport in amoebae
focused on passive diffusion across the nuclear envelope. A
comparison of the data obtained for amoebae, in which
polyvinylpyrrolidone-coated gold particles were used as a
transport substrate (Feldherr, 1965), with diffusion studies
performed on other cell types (Peters, 1986; Paine, 1992),
clearly indicate that both the rate of diffusion and the
dimensions of the diffusion channel are greater in amoebae
than in vertebrate cells. The present data show that there are
also significant differences in signal-mediated transport across
the envelope. In amoebae, the transport channels have a smaller
diameter, bipartite NLS is less effective in facilitating nuclear
import, and the nucleoporins do not appear to be glycosylated.
MATERIALS AND METHODS
Cell cultures
Amoeba proteus and the multinucleated amoeba Chaos carolinensis
were obtained from Carolina Biological Supply (Burlington, NC). The
cells were cultured at room temperature (21°C) in a medium
containing 0.5 mM CaCl2, 0.05 mM MgCl2, 0.16 mM K2HPO4, 0.12
mM KH2PO4 (pH 6.9-7.0), and were fed Tetrahymena pyriformis
(Prescott and James, 1955).
Preparation of BSA conjugates
The following peptides were synthesized by the University
of Florida protein core facility: CGGGPKKKRKVGG;
CGGGAVKRPAATKKAGQAKKKKLNGG; CGGGPKNKRKVGG;
CGGGVKRKKKPGG. They contain, respectively, the SV40 large T
NLS (a simple signal), the nucleoplasmin NLS (a bipartite signal), an
inactive mutant form of the large T NLS (cT mutant; Lanford and
Butel, 1984) and the reverse large T NLS. The signal domains are
underlined. The synthetic peptides were conjugated to bovine serum
albumin (BSA; Sigma, St Louis, MO) using the cross-linker mmaleimidobenzoyl-N-hydroxysuccinimide ester (Pierce, Rockford,
IL), as outlined by Lanford et al. (1986). Based on SDS-PAGE
analysis, it was estimated that an average of 8 peptides were
conjugated to each BSA molecule.
Fluorescent conjugates were prepared as described above, except
that the BSA was labeled with fluorescein before it was conjugated
with the signal peptides. Fluorescent labeling was accomplished by
treating 10 mg/ml BSA in 100 mM carbonate buffer (pH 9.0) for 1
hour with 2.25 mM fluorescein isothiocyanate (Pierce; Rockford, IL)
dissolved in 60 µl dimethylformamide (Pierce; Rockford, IL). The
labeled protein was purified by gel filtration.
Preparation of colloidal gold
Colloidal particles, ranging in diameter from 20 to 180 Å, were
prepared by reducing chloroauric acid with a saturated solution of
phosphorous in ether (Feldherr, 1965). A second fraction, containing
particles up to 290 Å in diameter, was prepared by using sodium citrate
as a reducing agent (Frens, 1973). These fractions, designated
intermediate and large, respectively, were coated with BSA conjugates
as described by Dworetzky et al. (1988). It is estimated that the protein
coat increases the diameter of the particles by approximately 30 Å.
Prior to microinjection, the coated gold preparations were concentrated
about 200-fold using Centriprep and Microcon concentrators (Amicon,
Inc., Beverly, MA), and dialyzed against amoebae medium.
Microinjection, EM, and fluorescent analysis
Microinjections were performed at room temperature using an
inverted microscope and a hydraulic micromanipulator (Narishige
USA, Inc., Greenvale, NY). The amoebae were immobilized in an oil
chamber and injected with pipettes that had tip diameters of about 2
µm. Approximately 5-10% of the cell volume was injected.
For EM analysis, the amoebae were fixed in 4% glutaraldehyde for
30 minutes, postfixed in 2% osmium tetroxide for 30 minutes,
embedded in Spurr’s resin, sectioned and examined with a JEOL
100CX electron microscope.
Kinetic analysis of the nuclear import of the fluorescein-labeled
conjugates was performed using an intensified Hamamatsu CCD
camera and a MetaMorph imaging system (Universal Imaging
Corporation, West Chester, PA). At the specified times after injection,
images were acquired and N/C fluorescent ratios were determined by
analyzing equal areas of nucleoplasm and cytoplasm. To minimize the
effects of UV on the cells, each exposure was limited to less than 1
second, and a number 16 neutral density filter was used.
RESULTS
To monitor nuclear transport, different size gold particles coated
with BSA conjugated to either simple, bipartite or mutant NLSs
were microinjected into the cytoplasm of amoebae, and their
subsequent nucleocytoplasmic distribution was analyzed by
electron microscopy. The relative rates of nuclear import are
expressed as nuclear/cytoplasmic (N/C) gold ratios, which were
determined by counting particles in equal, randomly selected
areas of nucleoplasm and cytoplasm. The functional size of the
transport channel was established by measuring the diameters
of the particles that entered the nucleus.
Table 1 shows the nucleocytoplasmic distribution obtained in
A. proteus for 20-180 Å gold particles coated with BSA
conjugates. The large T antigen NLS (Table 1A) facilitated the
transport of gold into the nucleoplasm. The N/C ratio reached
a value of 3.37 at 20 hours; however, there was restricted import
of particles larger than 100 Å in diameter (not including the
protein coat) and, as a consequence, the overall size distribution
of the particles present in the nucleoplasm was significantly less
than in the cytoplasm (Table 1E; P<0.00001; chi square test).
Table 1B shows that conjugates containing the bipartite,
nucleoplasmin NLS had only a limited capacity to facilitate
nuclear import. No uptake was detected at 30 minutes, and the
N/C ratios at 90 minutes and 20 hours were approximately 47
and 5.5 times lower, respectively, than those obtained for the
large T NLS conjugates. In addition, the size distribution of the
particles in the nucleoplasm was significantly less than that
observed for the simple NLS (P<0.00001). Gold particles
coated with the cT mutant (Table 1C) or reverse large T NLS
conjugates (Table 1D) were excluded from the nucleus.
To verify that there was limited nuclear uptake of particles
over 100 Å in diameter, experiments were performed using a
gold fraction in which the majority of the particles (>96%) had
diameters exceeding 120 Å. Fractions of this size (approximately
80-290 Å in diameter) have been used previously to investigate
Nuclear transport in the amoeba 2045
Table 1. Nuclear import in Amoeba proteus
% size distribution of particles (Å)‡
Experiment
Number of particles
measured
A BSA-large T NLS
30 min (6)*
90 min (6)
20 hours (7)
B BSA-nucleoplasmin NLS
30 min (6)
90 min (7)
20 hours (6)
C BSA-cT mutant NLS
30 min (6)
90 min (7)
20 hours (7)
D BSA-reverse large T NLS
30 min (4)
90 min (2)
20 hours (4)
E Cytoplasmic gold
20-40
40-60
60-80
80-100
100-120
120-140
140+
N/C ratio
mean (counts)
398
543
308
4.3
0.0
5.8
9.0
6.1
8.8
31.9
36.6
32.1
35.9
42.0
35.4
12.8
12.2
12.0
3.5
2.0
4.5
2.6
1.1
1.3
0.51 (496)
2.86 (285)
3.37 (491)
−
−
383
−
−
11.0
−
−
22.5
−
−
35.8
−
−
23.5
−
−
5.5
−
−
1.8
−
−
0.0
0.00 (233)
0.06 (222)
0.61 (232)
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0.00 (260)
0.00 (231)
0.005 (195)
−
−
−
367
−
−
−
1.6
−
−
−
3.5
−
−
−
22.1
−
−
−
40.3
−
−
−
21.5
−
−
−
7.4
−
−
−
3.5
0.00 (460)
0.00 (202)
0.00 (427)
*The number of cells injected is given in parentheses.
‡The data are shown as the % of different size particles present in the nucleus, except for ‘Cytoplasmic gold’, which shows the dimensions of the cytoplasmic
particles available for transport. Particle measurements do not include the protein coat.
the properties of the nuclear pores (Feldherr and Akin, 1995b).
In vertebrate cells, the N/C gold ratios obtained for these
fractions varied from about 1-3, and the largest particles able to
enter the nuclei were approximately 200-220 Å in diameter (not
including the protein coat). Furthermore, under normal growth
conditions, the size distribution of particles smaller than 200 Å
was essentially the same in both the nucleus and cytoplasm,
demonstrating that particles in this size range readily enter the
nucleoplasm. When large gold particles coated with BSA-large
T NLSs were microinjected into A. proteus, a very different
nucleocytoplasmic distribution pattern was observed (see Table
2). The N/C ratio was 0.05, demonstrating that the large particles
were essentially excluded from the nucleus. Furthermore,
measurements of the few particles that did enter the nucleoplasm
showed that only 40% were larger than 100 Å, whereas 98% of
the cytoplasmic particles (gold available for transport) were in
this size category. These results provide convincing evidence
that the functional size of the nuclear transport channel in
amoebae is significantly smaller than in vertebrate cells.
The nuclear import data obtained for C. carolinensis, with
regard to both N/C ratios and size distribution, were similar to
those described for A. proteus. In short-term experiments (30
minutes), there was facilitated uptake of gold particles coated
with BSA-large T NLSs (Table 3A). The nucleoplasmin signal
was much less effective in mediating transport (Table 3B), and
both the cT mutant (Table 3C) and reverse signals (Table 3D)
were excluded from the nucleus. The size distribution of
particles in the cytoplasm are shown in Table 3E. The main
difference in the C. carolinensis results was the finding that
essentially no gold particles coated with BSA-large T NLS
conjugates were present in either the nucleus or cytoplasm in
20 hour experiments (Table 3A). Electron microscopic analysis
of the amoebae revealed that particles coated with BSA-large
T NLSs, but not other signals, are sequestered from the
cytoplasm into food vacuoles (Fig. 1A-D). The fact that the
gold count in the nucleoplasm also drops, demonstrates that
particles are able to reenter the cytoplasm.
Since the effectiveness of a specific NLS could be a function
of the molecular size of the transport substrate, differences in
NLS activity were also investigated using fluorescein-labeled
BSA conjugates. The nuclear import kinetics of conjugates
containing either large T antigen, nucleoplasmin or cT mutant
NLSs, as well as labeled BSA alone, were determined in A.
proteus by measuring the N/C fluorescence ratio at intervals of
5, 10, 15, 30 and 60 minutes after injection. The results are
given in Fig. 2. Tracers containing the large T antigen or the
nucleoplasmin NLS concentrated in the nuclei during the course
of the experiments; however, the large T conjugates were
transported at a significantly greater rate (P<0.0001, ANOVA).
Interestingly, the nucleoplasmin signal was more effective in
Table 2. Nuclear import of large gold particles in Amoeba proteus
% size distribution of particles (Å)‡
Experiment
BSA-large T NLS
90 min (4)*
Cytoplasmic gold
Number of particles
measured
<80
80-100
100-120
212
427
27.4
1.2
32.5
0.9
18.4
0.9
120-140 140-160
9.0
12.2
5.2
16.9
160-180
180-200
200-220
220+
5.2
16.2
1.4
20.8
0.9
16.4
0.0
14.4
N/C ratio
mean (counts)
0.05 (1017)
*The number of cells injected is given in parentheses.
‡The data are shown as the % of different size particles present in the nucleus, except for ‘Cytoplasmic gold’, which shows the dimensions of the cytoplasmic
particles available for transport. Particle measurements do not include the protein coat.
2046 C. M. Feldherr and D. Akin
Fig. 1. Electron micrographs showing the fate of gold particles coated with BSA conjugates following injection into the cytoplasm of C.
carolinensis. At 30 minutes, gold particles (arrowheads) coated with BSA-large T NLSs were present in both the cytoplasm and the
nucleoplasm (A). By 20 hours, the BSA-large T NLS-gold (arrowhead) was found in food vacuoles (B), but was no longer present in either
the cytoplasm or nucleoplasm (C). Gold particles coated with BSA-cT mutant signals were still located in the cytoplasm after 20 hours (D).
c, cytoplasm; n, nucleus; fv, food vacuole.
mediating the transport of fluorescent conjugates than the larger
colloidal gold particles. The BSA-cT mutant NLS, and BSA
alone showed low levels of nuclear associated fluorescence. In
both instances, the N/C ratios were less than 1, and were not
significantly different. Since these ratios did not increase with
time, it is most likely that the nuclear fluorescence observed for
the two substrates that lacked an active NLS actually represents
background from overlapping and underlying cytoplasm.
The effect of wheat germ agglutinin (WGA) on nuclear
import in A. proteus is shown in Table 4. WGA, which binds
GlcNAc, blocks signal-mediated transport in vertebrate cells
when injected at a concentration of 3 mg/ml (Daubauvalle et
al., 1988). In amoebae, the same concentration of WGA had
no effect on the nuclear uptake of gold particles coated with
BSA-large T NLS conjugates. Both the N/C ratios and the size
of the particles that entered the nucleus were not significantly
different from controls. In a positive control, performed on
3T3-BALB/c cells, 3 mg/ml WGA completely blocked the
nuclear import of gold (data not shown).
As is the case in vertebrate cells, nuclear transport in
Table 3. Nuclear import in Chaos carolinensis
% size distribution of particles (Å)‡
Experiment
Number of particles
measured
A BSA-large T NLS
30 min (5)*
20 hours (6)
B BSA-nucleoplasm NLS
30 min (6)
20 hours (5)
C BSA-cT mutant NLS
30 min (6)
20 hours (5)
D BSA-reverse large T NLS
30 min (4)
20 hours (4)
E Cytoplasmic gold
20-40
40-60
60-80
80-100
100-120
120-140
140+
N/C ratio
mean (counts)
460
−
0.0
−
3.0
−
37.6
−
43.5
−
12.4
−
2.8
−
0.6
−
0.60 (359)
0.00 (0)
−
291
−
3.1
−
20.3
−
59.5
−
14.4
−
2.7
−
0.0
−
0.0
0.01 (126)
0.32 (150)
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0.00 (165)
0.00 (148)
−
−
367
−
−
1.6
−
−
3.5
−
−
22.1
−
−
40.3
−
−
21.5
−
−
7.4
−
−
3.5
0.00 (324)
0.004 (958)
*The number of cells injected is given in parentheses.
‡The data are shown as the % of different size particles present in the nucleus, except for ‘Cytoplasmic gold’, which shows the dimensions of the cytoplasmic
particles available for transport. Particle measurements do not include the protein coat.
Nuclear transport in the amoeba 2047
subunits (presumably the largest particles that cross the nuclear
envelope). There is evidence that nascent ribosomal subunits
in amoebae are contained in extended helical structures. These
structures can range from 300-800 Å in width, and up to 7000
Å in length; however, they are formed by the supercoiling of
filaments that are only 120-130 Å in diameter (Stevens, 1967).
Furthermore, there is morphological data suggesting that the
filaments exit the nucleus through the pores and dissociate once
in the cytoplasm (Stevens and Prescott, 1965). Thus, it is likely
that a functional pore diameter, of the order of 130 Å, is
sufficient to satisfy the transport requirements in amoebae, but
would not be adequate in vertebrate cells, where the ribosomal
subunits that exit the nucleus are much larger.
Although the simple and bipartite NLSs are both recognized
by the transport machinery in amoebae, the bipartite signal was
significantly less effective in the transport of both fluoresceinlabeled BSA conjugates and colloidal gold particles; furthermore,
its relative activity was found to vary inversely in relation to the
size of the transport substrate. The fact that the bipartite NLS is
more active in vertebrate cells could be a response to more
complex regulatory requirements in higher organisms, and might
be related to evolutionary changes in importin α. Recent analysis
of the crystal structure of importin α (Conti et al., 1998)
demonstrated that it contains two NLS binding sites. One site can
accommodate up to five lysine and/or arginine residues, whereas
the second, smaller site is able to bind only two basic residues.
Furthermore, the positioning of the two binding sites is ideally
suited for interactions with bipartite signals. If amoebae contain
a homolog of importin α that lacks the smaller NLS binding
domain, it would still be effective in transporting simple signals
but, due to lower binding affinity, would be less active in
mediating the transport of bipartite signals.
A number of vertebrate nucleoporins contain O-linked
GlcNAc (Pante and Aebi, 1996). Consistent with this finding,
WGA, or antibodies against epitopes that include O-linked
GlcNAc, bind to the pore complex and block signal-mediated
Fig. 2. The nuclear import rates of fluorescein-labeled BSA alone
(䉱), and labeled BSA conjugated with either large T antigen (䉬),
nucleoplasmin (䊏) or cT mutant (䊉) NLSs. Respectively, 5, 6, 7 and
5 cells were analyzed for the different transport substrates. Bars
indicate s.e.m.
amoebae is temperature-dependent (Table 5). A decrease in
temperature from 21°C to 4°C prevented transport of gold
particles coated with the BSA-large T NLS conjugate.
DISCUSSION
It was found that (1) the size limit for signal-mediated transport
into the nucleus is significantly lower in amoebae than in
vertebrate cells; (2) both the simple and bipartite NLSs mediated
nuclear transport, but the simple signal was significantly more
effective; (3) WGA had no effect on nuclear import, indicating
that amoeba nucleoporins are not glycosylated; and (4) signalmediated import is temperature-dependent. Results were also
obtained suggesting that, in amoebae, the simple NLS can also
function in mediating nuclear export.
Differences in functional pore size could be related to the
way in which specific cells process and transport ribosomal
Table 4. The effect of 3 mg/ml WGA on nuclear transport in Amoeba proteus
% size distribution of particles (Å)‡
Experiment
Number of particles
measured
BSA-large T NLS+WGA (9)*
BSA-large T NLS (9)
Cytoplasmic gold
379
348
573
20-40
40-60
60-80
80-100
100-120
120-140
140+
0.0
0.0
0.0
7.4
6.9
2.1
33.8
28.7
14.8
49.6
50.3
37.5
6.3
10.3
30.7
2.6
2.9
7.9
0.3
0.9
7.0
N/C ratio
mean (counts)
0.64 (422)
0.68 (314)
*The number of cells injected is given in parentheses.
‡The data are shown as the % of different size particles present in the nucleus, except for ‘Cytoplasmic gold’, which shows the dimensions of the cytoplasmic
particles available for transport. Particle measurements do not include the protein coat.
Table 5. The effect of temperature on nuclear transport in Amoeba proteus
% size distribution of particles (Å)‡
Experiment
Number of particles
measured
20-40
40-60
60-80
80-100
100-120
120-140
140+
N/C ratio
mean (counts)
0.00 (517)
0.00 (357)
0.42 (1134)
0.00 (423)
BSA-large T NLS+4°C (6)*
BSA+4°C (5)
BSA-large T NLS+21°C (8)
BSA+21°C (8)
340
-
0.0
-
9.7
-
28.5
-
45.0
-
14.1
-
2.6
-
0.0
-
Cytoplasmic gold
355
0.0
4.8
19.7
31.0
31.0
7.3
6.2
*The number of cells injected is given in parentheses.
‡The data are shown as the % of different size particles present in the nucleus, except for ‘Cytoplasmic gold’, which shows the dimensions of the cytoplasmic
particles available for transport. Particle measurements do not include the protein coat.
2048 C. M. Feldherr and D. Akin
transport at the translocation step. Based on the apparent absence
of glycoproteins in yeast and amoeba nucleoporins, it is unlikely
that GlcNAc is essential for translocation. Evidence favoring a
regulatory role for GlcNAc in higher organisms has recently been
reviewed by Snow and Hart (1998). GlcNAc moieties can be
readily added or removed from proteins, a dynamic feature that
would facilitate regulatory activities. Furthermore, there are
indications that O-linked glycosylation and phosphorylation can
occur at the same sites on proteins. Since there is evidence that
phosphorylation is involved in nuclear import (Vandromme et al.,
1994; Feldherr and Akin, 1995a; Mishra and Parnaik, 1995),
GlcNAc could affect transport by modulating the levels of
nucleoporin phosphorylation.
The sequestration of gold particles coated with active, but
not mutated NLSs into the food vacuoles of C. carolinensis not
only suggests a possible method of regulating the intracellular
levels of nuclear proteins in these primitive organisms, but also
represents a fortuitous experimental system for selectively
reducing the cytoplasmic concentration of nuclear import
substrates. In this study, when gold particles coated with BSAlarge T NLS conjugates were sequestered from the cytoplasm,
there was an accompanying loss of particles from the nucleus,
demonstrating that transport is reversible. Since the gold
particles used in these experiments are too large to diffuse
through the pores, as indicated by the fact that particles of the
same size coated with mutant signals are unable to enter the
nucleus (see Table 1C,D), the possibility exists that, in
amoebae, the simple NLS can also function as an export signal.
This dual role for the NLS is consistent with a facilitated
transport mechanism (Paine, 1993).
The data obtained in this report demonstrate that there are
significant differences in nuclear transport capacity in
organisms that presumably have different regulatory
requirements for nucleocytolasmic exchanges. In future
experiments it will be important to correlate these changes with
specific cellular activities; for example, the relationship
between functional pore size and ribosome production. From
an evolutionary standpoint, it is also important to understand
the differences that exist at the molecular level. In this regard,
it will be of interest to establish whether (1) Ran is necessary
for transport in amoebae (the fact that import is temperaturedependent suggests that energy is required); (2) xFxFG repeats
are present in amoebae nucleoporins, and function in
translocation, which seems to be the case in vertebrate cells;
and (3) different pathways are available for different classes of
proteins (e.g. ribosomal and hnRNP proteins).
We would like to thank Dr Robert Cohen for critically reviewing
the manuscript. This work was supported by grant MCB-9723015
from the National Science Foundation.
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