ABSTRACT
GTP-hydrolysis, the small ras-related GTP-binding protein Ran and its cognate guanosine nucleotide
exchange factor, the
RCC1
gene product, have recently been identified as essential components of the protein nuclear import pathway.
In this report we use three independent approaches to investigate the role of
these components in U1 snRNP nuclear import in somatic cells. (i) Using a
somatic cell based
in vitro
nuclear import system we show that U1 snRNP nuclear import, in marked contrast to protein transport, is not significantly inhibited by non-hydrolyzable GTP-analogs and is therefore unlikely to require GTP-hydrolysis. (ii) Using the dominant negative Ran mutant RanQ69L, which is
defective in GTP-hydrolysis, we show that Ran-mediated GTP-hydrolysis is not essential for the nuclear import of U1 snRNP
in microinjected cultured cells. (iii) Using a cell line expressing a
thermolabile
RCC1
gene product, we show that the nuclear accumulation of microinjected U1 snRNP
is not significantly affected by RCC1 depletion at the non-permissive temperature, indicating that RCC1 function is not essential for U-snRNP nuclear import. Based on these observations we conclude that
protein and U-snRNP nuclear import in somatic cells differ in their requirements for GTP-hydrolysis, and Ran or RCC1 function. Based on these results, the
substrates for nucleocytoplasmic exchange across the NPC can be divided into
two classes, those absolutely requiring Ran, including protein import and mRNA
export, and those for which Ran is not essential, including U-snRNP nuclear import, together with tRNA and U1 snRNA nuclear export.
The bi-directional movement of macromolecules between the cytoplasmic and nuclear
compartments occurs solely through the nuclear pore complexes (NPC) embedded in
the nuclear envelope (NE). Although many smaller molecules can passively
diffuse across the NPC, others, particularly those larger than 40 kDa are
translocated by signal mediated mechanisms. Both the recognition of the nuclear
localisation signals (NLS) by their cognate receptors and docking at the NE
precede the energy dependent movement through the nuclear pores (reviewed in
1
). NLS recognition and NE docking requires two proteins, denoted importin-[alpha] and importin-[beta] (also referred to as karyopherin-[alpha] and -[beta], respectively) (
2
-
6
). The importin-[alpha]/[beta]-karyophile complex is presumed to dock with proteins on
the cytoplasmic face of the NPC (
5
,
7
). Subsequent nuclear entry requires an additional protein complex containing at
least the two proteins p25 [Ran/TC4, ras-related nuclear protein (
8
,
9
), a small GTP-binding protein] and p10/NTF2, as well as GTP-hydrolysis (
10
-
15
).
The U1, U2, U4 and U5 small nuclear ribonucleoprotein particles (U-snRNPs), essential components of the splicing machinery, are assembled in
a complex sequence of events in both the nuclear and cytoplasmic compartments. The RNA components of these U-snRNPs are co-transcriptionally capped with a 7-methyl-guanosine-cap (m
7
G-cap) structure which constitutes part of their nuclear export signal (
16
). Once in the cytoplasm these U-snRNAs, which also share a single-stranded uridine rich sequence referred to as the Sm-binding site, are assembled into an RNA-protein complex, referred to as the Sm-core domain, which contains members of the Sm-protein family (
17
). The m
7
G-cap is then hypermethylated to a 2,2,7-trimethylguanosine-cap (m
3
G-cap), an event which effectively inactivates the nuclear export signal.
Additional RNP specific proteins are then added to such Sm-core particles prior to, during or after re-entry into the nucleus (reviewed in
18
).
From studies using microinjected
Xenopus
oocytes, we know that these U-snRNPs possess a complex bipartite nuclear localisation signal (NLS)
composed of the Sm-core domain and the m
3
G-cap (
19
-
22
). However, based on recent results obtained using both microinjected cultured
cells and an
in vitro
snRNP nuclear import system, it is now clear that the Sm-core domain alone is both necessary and sufficient to mediate the nuclear
targeting of U-snRNPs in somatic cells (
23
,
24
). The reported m
3
G-cap dependence is a characteristic unique to oocytes (
24
). U-snRNP nuclear import has the following features (
23
): (i) it is ATP and temperature dependent, (ii) it is a saturable process, and (iii) it requires soluble cytosolic factors. At least some components of the U-snRNP and protein nuclear import pathway differ since these two karyophile
classes do not cross compete for limiting cytosolic factors. Nevertheless, both
U-snRNPs and proteins enter the nucleus through the same or structurally
similar nuclear pores, since antibodies directed against NPC proteins inhibit
the transport of both karyophile classes. However, the two karyophile classes
appear to interact differently with the NPC during translocation from the
cytoplasm to the nucleus, since wheat germ agglutinin (WGA) has only a limited
inhibitory effect on U-snRNP nuclear accumulation under conditions which completely abolish protein import (
23
,
25
).
The recent observation that protein nuclear import requires Ran-mediated GTP-hydrolysis has lead to the suggestion that such small ras-related GTP-binding proteins are general components of the nucleocytoplasmic transport machinery (
11
-
13
,
15
). GTP-binding proteins are a super family of proteins known to function as
molecular switches in diverse cellular events including vesicle targeting (
26
), protein synthesis and targeting to the ER by the signal recognition particle
(
27
), as well as mRNA export (
28
-
30
). Ran (M
r
~25 kDa) is a predominately nuclear protein representing ~0.36% of total HeLa cell protein (
31
). At ~10
7
copies/cell Ran is ~20-fold more abundant than its cognate guanosine nucleotide exchange
factor, RCC1, and its GTPase activating protein, RanGAP1 (
32
). The phenotypes associated with mutations in Ran or Ran-interacting proteins are pleiotropic and relate to diverse cellular
functions which include cell cycle progression, nuclear structure, RNA
processing and export as well as protein import (reviewed in
33
,
34
-
36
). Whereas in most cases it remains to be established whether these effects are the direct or indirect consequence of Ran or Ran-interacting protein dysfunctions, in protein nuclear import, the direct
participation of Ran-mediated GTP-hydrolysis has been demonstrated (
11
,
13
,
15
,
35
). Recently, Ran-mediated GTP-hydrolysis has been mapped to an early step in protein nuclear
import observed exclusively on the cytoplasmic face of the NPC (
36
).
One mutation affecting a Ran-interacting protein involves the evolutionarily conserved
RCC1
(regulator of chromosome condensation 1) gene which was initially reported as the mutant gene responsible
for the pleiotropic temperature sensitive phenotypes of the tsBN2 cell line
(reviewed in
33
). Recently
RCC1
was implicated in RNA export (
28
-
30
) and protein import (
37
,
38
). Incubation of tsBN2 cells at the restrictive temperature leads to the rapid
degradation of the
RCC1
gene product (RCC1). At this time RCC1 is no longer detectable using
immunological assays (
31
,
37
,
39
). Loss of RCC1 function can be relieved by the re-introduction of wild type RCC1 (
37
) or either GDP- or GTP-bound Ran (
38
).
In this report we present
in vitro
and
in vivo
evidence that protein and U-snRNP nuclear import in somatic cells differ in their requirement for GTP-hydrolysis, RCC1 and Ran. These results suggest that the early steps
of the protein and U-snRNP nuclear import pathways differ, but do not exclude the possibility
that the two pathways converge at a later step prior to or at the site of the
actual NPC translocation.
SV40 T-antigen was purified as described (
41
). Recombinant human Ran and RanQ69L were expressed in
Escherichia coli
and purified as described (
38
). After loading with GTP, the identity of the nucleotide bound to the
recombinant protein was confirmed by HPLC (
42
).
HeLa and tsBN2 cells (
43
) were grown in Dulbecco's modified Eagle medium (Gibco, Eggenstein, Germany)
supplemented with antibiotics and 10% fetal calf serum (FCS) (BioChrom, Berlin,
Germany) in a humidified incubator at 33.5oC (tsBN2, permissive temperature) or 37oC (HeLa) under 10% atmospheric CO
2
.
For microinjection experiments, cells were plated at least 36 h before microinjection on glass coverslips (Eppendorf, Hamburg, Germany). An Eppendorf microinjector (model 5242/5170) mounted on an IM35
inverted microscope (Carl Zeiss, Oberkochen, Germany) was used to deliver
samples. The karyophiles, SV40 T-antigen (0.5 mg/ml), and U1 snRNP (1 mg/ml), were centrifuged for 15 min
at 14 000
g
, prior to microinjection. For co-injections, karyophile was mixed 1:1 with recombinant Ran (4 mg/ml). The
volume injected was estimated to be 5-10 * 10
-15
litres. Microinjection needles were pulled from glass capillaries on an
automatic pipette puller (David Kopf Instruments, Tujunga, USA).
Microinjected cells were washed three times with PBS (pH 7.4), fixed in 4% ice
cold paraformaldehyde in PBS for 15 min, permeabilized for 20 min in 0.2%
Triton X-100 in PBS, and blocked for at least for 1 h in 10% FCS in PBS to reduce non-specific staining. T-antigen was visualised by staining for 1 h with an antibody mix containing mouse monoclonal antibodies
Pab 101, Pab 221, Pab 416 and Pab 419 (10 [mu]g/ml each) (
38
) followed by FITC-conjugated second antibody (Jackson Immuno- Research Laboratories, West Grove, USA) (1:50) for 1 h. Antibodies
were diluted with 10% FCS in PBS, the incubations were carried out at room
temperature. After each antibody incubation, cells were washed three times
quickly and then another three times for 10 min each. After the last wash step,
the coverslips were air-dried, mounted in 90% glycerol containing 0.1 mg/ml
p
-phenylenediamine, and viewed on an Axiovert 135 microscope (Carl Zeiss,
Oberkochen, Germany) using a 63* objective.
Fluorescently labelled BSA-NLS conjugates were prepared as described (
23
). As judged by SDS-PAGE, fluorescein-labelled conjugates do not contain free label and have 10-20 NLS peptides (peptide sequence, PKKKRKV
132
EDPYC) per BSA molecule. Fluorescently labelled dextran, M
r
70 kDa, was obtained from Sigma (Deisenhofen, Germany). Isolated HeLa U1 snRNPs
were purified and labelled as described (
23
) except the NHS (
N
-hydroxy-succinimidyl)-Cy3 dye was used for labelling (Biological Detection Systems Inc., Pittsburgh, USA). Labelled U1 snRNPs
were purified using Centricon C-100 units (Amicon) and dialysed for 6 h against T-buffer (molecular weight cut off, 8 kDa). The purity and integrity
of labelled particles was confirmed by SDS-PAGE and sedimentation analysis on 5-20% glycerol gradients in T-buffer at 260 000
g
at 4oC for 6 h, in a Beckmann TLS-55 rotor as described (
23
).
Unless indicated otherwise, nuclear import assays were performed essentially as described (
23
). Untreated reticulocyte lysate (Serva, Heidelberg, Germany), pre-dialysed in T-buffer, was typically 50% of the transport mix volume. Karyophiles
were added to give a working concentration of 0.1 mM. ATP depleted conditions
were obtained by pre-incubation of the transport mix, without ATP, phosphocreatine and creatinephosphokinase, for 30 min at 37oC in the presence of either 10 U/ml apyrase or 10 U hexokinase/10 mM glucose and then incubation of subsequent transport
assays at 4oC. Standard transport assays were incubated for 60 min at 37oC, before termination by washing the coverslips 3 * 5 min in ice-cold PBS (pH 7.4) followed by 15 min fixation in 4%
paraformaldehyde/PBS on ice. Coverslips were then washed for 5 min each in PBS
and PBS containing 50 ng/ml bisbenzimide DNA dye (Hoechst 33258), and finally
three times in PBS, before air drying and mounting. Nuclear fluorescence was
quantified using a Kappa video camera (Kappa Messtechnik, Germany) linked to a Quantimet 570 running with customised Leica Q570 Software
(Leica, Bensheim, Germany) as described (
23
). For each mean fluorescence value ~100 nuclei were measured in at least two independent experiments.
We have recently established an
in vitro
system which accurately reproduces U-snRNP nuclear import
in vivo
(
23
). The recent demonstration that GTP is required for protein nuclear import
suggested that GTP may also be needed for U-snRNP nuclear import. Since our previous studies were routinely performed
in the absence of exogenously added GTP we have tested whether the inclusion of
GTP has a stimulatory effect on U-snRNP nuclear import.
For these studies we have used fluorescently labelled HeLa U1 snRNPs as model
karyophile. Isolated 10-12S U1 snRNP preparations contained the common Sm proteins (E, F, G, D1,
D2, D3, B and B'), the specific A, C and 70k proteins and also ~1% contaminating U5 snRNP proteins. These U1 snRNPs were
fluorescently labelled via primary amine groups of proteins exposed on the
intact particles and purified from excess dye and any proteins which dissociate
during labelling by microfiltration on Centricon C-100 units and dialysis. After this procedure, all of the U1 snRNP proteins
(E, F, G, D1, D2, D3, C, A, B, B' and 70k) are labelled although the labelling of the 70k and B, B' proteins is reproducibly weaker (Fig.
1
A, lane A). To confirm that labelled U1 snRNPs are intact 10-12S RNPs, we sedimented such particles on 5-20% glycerol gradients and analysed gradient fractions using SDS-PAGE. Consistent with previous studies (
23
), labelled U1 snRNP were found in fractions 5 and 6 as 10-12S particles (Fig.
1
A). A very small (<1%) amount of free unlabelled protein, in fractions 1 and 2, migrating at 70
kDa, was also detectable with Coomassie blue staining but not with UV induced
fluorescence. For comparison Figure
1
B shows that free fluorescently labelled proteins (BSA-NLS conjugates) sedimented in fractions 1 and 2 on equivalent gradients. Therefore our labelled U1
snRNP preparations contain intact ribonucleoprotein particles and only very few free labelled proteins. Cytoplasmically injected labelled U1 snRNPs accumulate in the nuclei of microinjected cultured
cells incubated at 37oC but not at 4oC, indicating that this import is an active process (data not shown).
In contrast, free dye does not accumulate in nuclei either in the
in vitro
system or when microinjected into the cytoplasm of cultured cells (
23
). Several experiments performed
in vitro
and
in vivo
, demonstrate that labelled U-snRNPs are transported as such and do not undergo disassembly/reassembly
events (
22
,
23
). Whereas the U1 snRNP specific proteins are known to enter the nucleus by the
conventional protein import pathway (
18
), the free Sm core proteins are known to enter the nucleus only in the form of
RNPs (
18
).
Independent evidence supporting our earlier conclusion that GTP-binding proteins, in particular Ran, are not essential for U-snRNP nuclear import, was obtained using the mutant baby hamster
kidney cell line tsBN2 which expresses a temperature sensitive
RCC1
gene product (
43
). Incubation of these cells at the restrictive temperature leads to the rapid
degradation of the RCC1, thereby disrupting Ran GTP/GDP cycling and leading to
a defect in protein nuclear import (see Introduction) (
37
,
38
). If Ran GTP/GDP cycling is essential for U1 snRNP nuclear import, then RCC1
depletion might be predicted to lead to a U1 snRNP transport defect.
We therefore pre-incubated tsBN2 cells for 6 h at either the permissive (33.5oC) or restrictive (39.5oC) temperatures prior to cytoplasmic microinjection of
karyophile. After injection the incubation was continued at the same
temperature for 60 min prior to fixation and preparation for fluorescence-microscopy. After 6 h incubation at the non-permissive temperature, little or no RCC1 is detectable
immunologically (
31
,
37
-
39
). As seen in Figure
4
A, the observed nuclear accumulation of U1 snRNP was equally efficient at both
the permissive and restrictive temperatures (compare panels 3 and 4). In contrast, the nuclear import of the SV40 T-antigen was drastically inhibited at the restrictive, but not the
permissive, temperature (compare panels 1 and 2).
To quantitatively evaluate the ability of tsBN2 cells to import karyophile, a
kinetic analysis of karyophile nuclear accumulation was performed and the microinjected cells displaying predominantly nuclear or cytoplasmic signals were counted and expressed as a percent of the total number of stained cells. As shown in Figure
4
B, this quantitational analysis confirmed our interpretation of Figure
4
A. Consistent with our
in vitro
data, these results suggest that RCC1, and therefore indirectly also Ran, is
not essential for U1 snRNP nuclear import.
To directly test the possible involvement of Ran-mediated GTP-hydrolysis in U1 snRNP nuclear import, we have studied the effects
of co-injecting a dominant negative Ran mutant, designated RanQ69L, into
cultured cells. As a consequence of changing glutamate residue 69 into a
leucine, RanQ69L is GTPase deficient and therefore accumulates in the GTP-bound form (
31
,
32
,
44
). Thus, RanQ69L would be expected to induce the same phenotype as the addition
of non-hydrolyzable GTP-analogs. Indeed, RanQ69L dramatically inhibits protein nuclear import both
in vitro
(
36
) and
in vivo
(
38
), presumably by acting as a competitive inhibitor that non-productively binds to Ran interacting proteins, such as nuclear pore
components and RanGAP1.
Recombinant GTP-bound human Ran (Ran.GTP) and RanQ69L (RanQ69L.GTP), was prepared and
charged with GTP as described previously (
32
,
38
; data not shown). Based on HPLC analysis, >95% of nucleotide bound to these
recombinant proteins was GTP, the rest being GDP (data not shown). Both recombinant proteins display the expected functional phenotypes: the GTPase activity of the human Ran, but not the mutant RanQ69L, was
stimulated several orders of magnitude by RanGAP
in vitro
(
32
; data not shown).
As seen in Figure
5
A, co-injection of RanQ69L.GTP together with karyophile into tsBN2 cells
incubated at the permissive temperature resulted in a drastic inhibition of
protein transport (compare panels 1 and 3), but induced only a minimal effect
on the observed U1 snRNP nuclear import after a 60 min incubation (compare
panels 2 and 4). Control co-injections of recombinant wild-type Ran.GTP (data not shown) or GTP alone in buffer (panels 1 and
2) had no significant effect on either protein or U1 snRNP transport. We
estimate that the amount of injected recombinant Ran is equivalent to ~5% of total cellular Ran and is equimolar with cytoplasmic Ran (
32
).
Conceivably only weak effects of the mutant Ran, or even the RCC1 depletion, on
U1 snRNP transport could be missed by our assay. In contrast the combined
effects of RanQ69L.GTP co-injection and RCC1 depletion could be expected to have a more obvious
effect on U1 snRNP transport. We therefore also tested the effects of
RanQ69L.GTP co-injection into tsBN2 cells incubated at the restrictive temperature. As
shown in Figure
5
B the inhibitory effect on protein import, after 60 min incubation, was even
more drastic (compare panels 1 and 3) than with either treatment alone (Figs
4
A and
5
A), whereas U1 snRNP transport remained largely unperturbed (compare panels 2
and 4). A kinetic analysis of the co-injection experiments (Fig.
5
C) confirmed our interpretation that RanQ69L.GTP inhibits protein (graphs 1 and
2), but not U1 snRNP, nuclear import (graphs 3 and 4). Together with our
earlier results (Fig.
4
), these results indicate that neither RCC1 nor Ran-GDP/GTP cycling are essential for U1 snRNP transport.
Small GTP-binding proteins are essential components of many fundamental transport
pathways. The identification of the small GTP-binding protein Ran, as an essential component of the protein nuclear
import (
11
,
13
,
15
) and RNA export (
28
-
30
) machinery, provided further evidence that this class of proteins play crucial
roles in cellular targeting events. The recent report that tRNA and U1 snRNA
nuclear export is not dependent on RCC1 function (
40
) indirectly challenged the proposed universal role of Ran in nucleocytoplasmic
exchange.
Using three independent approaches, we provide
in vivo
and
in vitro
evidence that Ran-GDP/GTP cycling is not essential for U1 snRNP nuclear import in somatic cells: (i) using a homologous
in vitro
transport system supplemented with non-hydrolyzable GTP-analogs we show that GTP-hydrolysis is not essential for U1 snRNP nuclear import; (ii)
using the dominant negative Ran mutant, RanQ69L, which displays a defective GTP-hydrolysis, we provide
in vivo
evidence that Ran mediated GTP-hydrolysis is not essential for U1 snRNP nuclear import; and finally,
(iii) using the temperature sensitive cell line tsBN2 which expresses a
thermolabile
RCC1
gene product, the only known guanosine nucleotide exchange factor for Ran, we
show that RCC1 mediated exchange of Ran bound GDP with GTP
in vivo
is also not essential for U1 snRNP nuclear import. Our
in vitro
studies with the non-hydrolyzable GTP-analogs also argue against the possible involvement of a further, as
yet unidentified, GTP-binding protein in U1 snRNP nuclear import. In sum, our data argue against
a role for GTP-binding proteins, such as Ran, as universal regulators of
nucleocytoplasmic exchange (
11
-
13
,
33
). Instead our results, together with the observation that tRNA and U1 snRNA
nuclear export are RCC1-independent, and therefore probably also Ran-independent (
40
), suggest that at least two pathways exist for nucleocytoplasmic exchange, one
of which is absolutely Ran-dependent and the other which is not.
Available data suggest that at least some early components of the protein and U-snRNP nuclear import pathways differ. These include (i) their different
sensitivity to wheat germ agglutinin which binds to
N
-acetyl-glucosamine modified nuclear pore proteins, (ii) the inability of
these two karyophile classes to cross compete for limiting transport factors (
23
,
25
,
45
), and now also (iii) their differential Ran requirements. Despite these
differences, based on the ability of an antibody directed against the NPC
protein p62 to inhibit the nuclear import of both karyophile types, both
proteins and U-snRNPs are believed to be translocated through the same or structurally
similar NPCs (
23
,
25
). As shown schematically in Figure
6
, consistent with the concept of a common translocating machinery, p62 has been
localised to both the cytoplasmic and nuclear faces of the NPC (reviewed in
1
). Current data suggest that the initial energy independent docking of
karyophilic protein at the NPC occurs at sites some 60 nm from the central
plane, a region corresponding with the NPC-fibrils projecting into the cytoplasm (
36
,
46
, reviewed in
1
; Fig.
6
). This region also coincides with the sites of protein karyophile accumulation
induced by non-hydrolyzable GTP analogs or by the GTPase deficient Ran mutant, RanQ69L,
suggesting that at least one site of GTP-hydrolysis is at or very close to the initial docking site (
36
,
46
, reviewed in
1
; Fig.
6
). Conceivably additional sites of GTP-hydrolysis events, not detected in this study (
36
), could exist along the protein import pathway to, and through, the central
channel of the NPC.
The data presented in this study suggest that GTP-hydrolysis or Ran-GDP/GTP cycling is not essential for U1 snRNP nuclear import in
somatic cells. Therefore, if the protein and U-snRNP import pathways utilise common components at some point, these must
lie beyond the sites of transport arrest induced by Ran dysfunction, as
depicted in route 1 (Fig.
6
). Alternatively U-snRNPs may enter the import machinery via a completely different route,
involving an initial docking with U-snRNP specific NPC structures (Fig.
6
, route 2), or alternatively have an abbreviated import pathway and access the
common import machinery at a point downstream of the Ran-mediated checkpoint (Fig.
6
, route 3). In this context, based on our observation that fluorescently
labelled U-snRNP can accumulate at the NE under ATP-depletion conditions in a modified
in vitro
import assay (unpublished data), much as described for karyophilic proteins (
36
,
46
, reviewed in
1
), it will be interesting to compare the sites of karyophile accumulation under
these conditions at the ultrastructure level. Likewise, using available NPC
protein mutants and a yeast based U-snRNP
in vitro
nuclear import system, it should soon be possible to directly address the role
of specific NPC proteins in protein and U-snRNP nuclear import.
We thank A. Brunahl and M. Hauser for preparation of antibodies, A. Badouin and
M. Wick for technical assistance, and C. Cole for providing tsBN2 cells.
Support from the Deutsche Forschungsgemeinschaft (Fa 138/4-3 to E.F., SFB 286 to R.L., Po 152/8-2 to H.P.), the Vanderbilt University and the Fonds der Chemischen
Industrie is gratefully acknowledged.
REFERENCES
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