1
Max Delbrück Center for Molecular Medicine,
Robert-Rössle-Strasse 10, D-13122 Berlin,
Germany
2
Department of Biochemistry, Humboldt University,
Charité, Hessische Straße 3-4, D-10115
Berlin, Germany
3
Center for Biochemistry and Molecular Cell Chemistry, Georg-August University,
Heinrich-Düker-Weg 12, D-37073
Göttingen, Germany
4
Franz-Volhard-Klinik, Humboldt University,
Charité, Wiltbergstraße 50, D-13122
Berlin, Germany
*
Author for correspondence (e-mail:
brigitte.wiedmann{at}charite.de
Accepted April 6, 2001
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SUMMARY |
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Key words: NAC, Nuclear transport, Saccharomyces cerevisiae
![]() |
INTRODUCTION |
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Several functions have been attributed to NAC or its subunits. The entire
complex at the ribosome protects nascent polypeptides from premature
interactions with cytosolic proteins. The NAC complex also inhibits the
binding of ribosomes that translate non-secretory proteins to translocation
sites in the membrane of the endoplasmic reticulum (Wang et al.,
1995; Wiedmann et al.,
1994
; Lauring et al.,
1995a
; Lauring et al.,
1995b
;
Möller et al.,
1998
; Wiedmann and Prehn,
1999
). NAC is associated with
the ribosomes and binds to emerging nascent polypeptides before any other
cytosolic protein. NAC releases the nascent chain when a binding domain for
cytosolic proteins such as the signal recognition particle (SRP) or chaperones
is completed (Wang et al.,
1995
). Furthermore, NAC may
function in cotranslational protein import into mitochondria (George et al.,
1998
,
Fünfschilling and Rospert,
1999
).
In addition to the functions of ribosome-bound NAC in the cytosol, single
NAC subunits may interact with DNA thereby regulating transcription (Parthun
et al., 1992; Moreau et al.,
1998
). Such a function would
imply that at least some NAC protein is located transiently within the nucleus
and that the NAC subunits contain signals for their active translocation into
the nucleus. Indeed, the NAC
-subunit has been found in the nuclei of
serum-deprived osteoblastic cells in mice (Yotov, et al.,
1998
). However, verification
of this finding has been difficult (Beatrix et al.,
2000
). NAC subunit
translocation into nuclei should require import receptors. According to our
present knowledge, nuclear import substrates bind to soluble receptors of the
importin ß family either directly or with the help of specific adapter
proteins (importin
) and translocate through the nuclear pore driven by
the concentration gradient of Ran-GTP (Wozniak et al.,
1998
;
Görlich and Kutay,
1999
). Most presently known
substrates bear a so called `classical' nuclear localization signal (NLS).
These proteins interact with their receptor importin ß via adapter
molecules of the importin
family. Other substrates contain a NLS which
allows their direct interaction with one of the various import receptors. Some
proteins need a dimer of two different importins for efficient transport. For
example, histone H1 requires importin ß and importin 7
(Jäkel et al.,
1999
). Other proteins can use
more than one import pathway due to their ability to bind to different import
receptors. The yeast ribosomal protein L25 is imported by Yrb4p (also known as
Kap123p) and Pse1p (also known as Kap121p) (Rout et al.,
1997
; Schlenstedt et al.,
1997
; Seedorf and Silver,
1997
). The human homologue,
L23a, can even be imported by four different receptors, namely importin 5,
importin 7, importin ß, and transportin
(Jäkel and
Görlich,
1998
). We report here, that
non-ribosome-associated yeast NAC subunits can translocate into the nucleus in
vivo. Using in vitro assays and in vivo experiments, we demonstrate that this
transport is specific and can be mediated by several of the known import
factors.
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MATERIALS AND METHODS |
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GFP fusion plasmids of EGD1 and EGD2 were constructed by
cloning 500 base pairs upstream of ATG and the coding region in frame to two
C-terminal copies of modified GFP in pRS414 (Sikorski and Hieter,
1989; Cormack et al.,
1997
). To yield truncated
versions of EGD1 the promoter region of EGD1 was cloned via
a ClaI restriction site to the coding region of EGD1 without
its N-terminal 33 (
N11-egd1), 42 (
N14-egd1),
81 (
N27-egd1) or 132 (
N44-egd1) base pairs
into pRS415, which already contained the EGD2 gene. The new ATG start
codon was introduced with oligonucleotides that were designed to fuse the
promoter with the shortened coding region. These mutated genes were cloned via
SacI and EcoRI restriction sites in frame into pRS414-2GFP
in order to create GFP fusion proteins. The plasmid pRS414-2GFP was a gift of
U. Lenk (MDC, Berlin). All constructs were sequenced, and the expression of
fusion proteins was verified by SDS/PAGE and western blot analysis.
Recombinant expression of proteins
Coding regions of EGD1 and EGD2 were cloned via PCR into
BamHI and HindIII sites of pQE 30 vector (Qiagen).
Expression of the His-tagged proteins was induced by IPTG in Escherichia
coli M15 at 25°C for 4 hours. HIS-Egd2p was purified under native
conditions according to the QIAexpress protocol. Briefly, harvested cells of a
1 litre culture were resuspended in 100 mM sodiumphosphate buffer, pH 7.8, 300
mM NaCl and lysed by freeze/thawing and sonication. The lysate was centrifuged
(30 minutes, 10,000 g) and the supernatant (OD600
1) was loaded onto a pre-equilibrated Ni-NTA-column (8 ml 50%
Ni-NTA-resin). The protein was eluted by 50 mM Hepes, pH 3.5 and neutralized
immediately. His-Egd1p was purified under denaturing conditions according to
the QIAexpress protocol. The difference to the His-Egd2p purification was that
the cells were resuspended in 100 mM sodiumphosphate buffer, pH 8.0, 8 M urea.
His-Egd1p was renatured bound to the Ni-NTA-resin by a gradient of 8-0 M urea
in 100 mM sodiumphosphate buffer, pH 8.0 and then eluted as described
above.
Preparation of recombinant import factors occurred as described earlier: C-
terminal His-tagged importin 1/Rch1 and Srp1p
(Görlich et al.,
1995
),
3,
4,
5/hSrp1 and
7 (Köhler et al.,
1999
), importin ß,
nucleoplasmin, nucleoplasmin core, human Ran, Schizosaccharomyces
pombe Rna1p, murine RanBP1 and NTF2 (Kutay et al.,
1997
). Expression clones for
importin ß, nucleoplasmin, nucleoplasmin core and NTF2 and recombinant
protein of human Ran, Schizosaccharomyces pombe Rna1p and murine
RanBP1 were kindly provided by D. Görlich (ZMBH,
Heidelberg).
In vitro nuclear protein import assay
Import assays were performed as described previously (Adam et al.,
1990;
Jäkel and Görlich,
1998
,
Köhler et al.,
1999
). Briefly, HeLa cells
were grown on three-well microscopy slides (Roth) to 40-80% confluency, washed
once in ice-cold PBS, and permeabilized for 8 minutes in ice-cold 20 mM
Hepes-KOH (pH 7.5), 150 mM potassium acetate, 5 mM magnesium acetate, 0.5 mM
EGTA, 250 mM sucrose, 30 µg digitonin (Sigma) per ml. The cells were
incubated for 8 minutes on slides with 20 µl of import mixture at room
temperature. The import reaction was stopped by fixation with 3%
paraformaldehyde/PBS. After washing in PBS, the slides were mounted with
Vectorshield mounting medium (Vector). Import mixtures contained an energy
regenerating system (0.5 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, 50
µg creatine kinase/ml), core buffer (2 µg nucleoplasmin core/ml, 20 mM
Hepes-KOH (pH 7.5), 150 mM potassium acetate, 5 mM magnesium acetate, 250 mM
sucrose), 0.5 mM EGTA, 3 µM RanGDP, 0.2 µMRna1p, 0.3 µM RanBP1 and
0.4 µM NTF2. The assay was adjusted to 10% with reticulocyte lysate, and 1
µM importin ß and 2 µM of importin
were added.
Fluorescence labelling of recombinant proteins was performed with Texas red
or fluorescein 5'-maleimide as described previously (Kutay et al.,
1997). Fluorescence microscopy
was performed with a confocal microscope (MRC 1024, BioRAD) equipped with a
63x oil-immersion objective (Nikon Diaphot). Images were processed by
Adobe Photoshop.
Fluorescence microscopy of yeast cells
Cultures of exponentially growing yeast cells were incubated at 30°C
(or 25°C for temperature sensitive mutants). To visualize nuclei, Hoechst
33258 (Sigma) was added to a final concentration of 10 µM for 10 minutes
prior to microscopy. Fluorescence microscopy was performed with living cells
using a Zeiss Axioplan 1 microscope equipped with a NEOFLUAR 100x/1.30
oil-immersion objective lens (Zeiss). GFP fluorescence was obtained using the
FITC channel. Stained nuclei were visualized in the UV channel. Photographs
were taken with a CCD camera (PCP computer optics, Kelheim) and the Axiovision
1 software (Zeiss). Images were processed by Adobe Photoshop.
Cell fractionation
Cells were grown to middle log-phase on SD, spheroplasted with Zymolyase
100T, homogenized in IP-buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM
Mg(OAc)2, 1 mM PMSF, and protease inhibitor mix (Sigma)) at about
0.5-1 g fresh weight/ml buffer in a Brown-homogenizer (Brown, Melsungen,
Germany). Cell debris, left over cells and nuclei were sedimented by two 15
minute centrifugation steps at 3,000 g. The supernatant was
cleared of mitochondria by a 15 minute centrifugation at 10,000
g. A 30 minute centrifugation at 400,000 g
yielded a supernatant free of endoplasmic reticulum. Ribosomes were separated
from cytosol by sedimentation at 400,000 g in a Beckman
TL100.4 rotor for 30 minutes. The volume of fractionation samples was adjusted
so that it equalled the original volume and electrophoresis lanes were
directly comparable.
Co-immunoprecipitation
The 40,000 g supernatant of a cell fractionation was used
for immunoprecipitation. 2 ml were incubated 2-4 hours with 3 µl antibody
against Egd1p and/or Egd2p at 6°C on a roller (antibodies provided by M.
Wiedmann, MSKCC, New York). Antigen-antibody complexes were collected with 50
µl 50% protein A-sepharose in IP-buffer, washed extensively with IP-buffer,
and solubilized by boiling in 50 µl 2x SDS sample buffer. Proteins
were separated on 12% SDS polyacrylamide gels. Western blot detection was
performed using the ECL system according to the instructions of the
manufacturer (Amersham Pharmacia Biotech).
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RESULTS |
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Next, we tested the location of Egd1p-2GFP. As expected from our cell
fractionation experiments, full length Egd1p-2GFP was found exclusively in the
cytosol in wild-type cells and also in the NAC-knockout strain
(Fig. 1B). Therefore, we
searched for EGD1 mutants that are deficient in ribosome binding and
shortened the coding region subsequently at its N-terminus. Removal of the
first 11 N-terminal amino acids (N11-Egd1p-2GFP) abolished the
association with ribosomes. Only traces of truncated Egd1p-2GFP were left in
the ribosomal fraction (Fig.
2A). The interaction of truncated Egd1p versions with the Egd2p
was not affected, as shown by co-immunoprecipitation
(Fig. 2B). The N-terminal
shortened Egd1p-2GFP accumulated in the nucleus of NAC-knockout cells (compare
Fig. 1B,C) and wild-type cells
(not shown). In summary, these experiments proved the ability of both proteins
(Egd1p, Egd2p) to enter the nucleus.
|
The in vitro import of yeast NAC into nuclei is energy dependent and
mediated by different importin proteins
We next investigated the nuclear import of these proteins in more detail.
First, we tested whether or not yeast NAC proteins can be transported into the
nucleus via the `classical' importin /importin ß-mediated import
pathway using an in vitro import assay (Adam et al.,
1990
;
Jäkel and Görlich,
1998
,
Köhler et al.,
1999
). Basically, HeLa cells
were permeabilized and the cytosol was washed out. Fluorescein labelled
recombinant Egd1p or Egd2p and the components necessary for proper import
including different human
-importins and the
-importin homologue
of yeast Srp1p were added to a standard in vitro import reaction. The
fluorescence signal in the nuclei of the samples containing importins
1/Rch1,
3,
7 or yeast
-importin Srp1p demonstrates
that both Egd2p and Egd1p were actively transported into nuclei
(Fig. 3a,b,d,e,m,n,p,q). The
human ß-importin alone (Fig.
3s,t) could not mediate transport of yeast NAC proteins. Several
lines of evidence indicate that the import of the yeast NAC subunits is due to
a specific binding to their import receptors. First, not all
-importins
could promote nuclear import of the yeast proteins. Importins
4 and
5 failed to import Egd1p or Egd2p
(Fig. 3g,h,j,k) even though
they could transport nucleoplasmin, a typical import substrate
(Fig. 3i,l). Second,
nucleoplasmin competed with the Egd2p for interaction with importin
1
(see Fig. 4), indicating that
the import of Egd2p requires an empty NLS-binding site on its import adapter
importin
1. Furthermore, there was no transport in the absence of
energy (Fig. 4i). Together,
these experiments show that yeast NAC subunits in vitro can enter the nuclei
of human tissue culture cells with components of the `classical' import
pathway.
|
|
In vivo transport of Egd1p into nuclei is inhibited in nuclear
protein import mutants
We then asked how yeast NAC proteins are imported in vivo and searched for
import mutants that could decrease the intense nuclear accumulation of
N11-Egd1p-2GFP. To evaluate the role of the importin
/importin
ß-mediated transport, we looked for localization of
N11-Egd1p-2GFP
in mutants of SRP1: srp1-31 and srp1-49 (Loeb et
al., 1995
; Shulga et al.,
1996
, Tabb et al.,
2000
). We observed no effect
in srp1-49 cells but a slight increase of cytosolic staining in
srp1-31 cells (Fig.
5A) after a 3 hour shift to the nonpermissive temperature. The
relatively small effect can be explained by the already existing strong
nuclear accumulation of
N11-Egd1p-2GFP at permissive temperature.
Another possible reason could be that other factors participate in Egd1p
nuclear import. NAC proteins are similar to ribosomal proteins in size and
expression level, and function in alliance with ribosomes in the cytosol. We
therefore focused on Kap123p and Pse1p, factors involved in the import of
ribosomal proteins (Rout et al.,
1997
; Schlenstedt et al.,
1997
; Seedorf and Silver,
1997
; Stage-Zimmermann et al.,
2000
). We tested whether or
not
N11-Egd1p-2GFP can accumulate in nuclei of
kap123
or pse1-1 mutants, as seen in the corresponding wild-type cells
(Fig. 5B). Both mutants showed
an enhanced fluorescence in the cytosol and a reduced fluorescence in the
nucleus (Fig. 5B, bottom four
images) compared with wild-type cells (Fig.
5B, top two images). The defective import into nuclei of
pse1-1 cells was already detectable at permissive temperature. We
conclude that the ribosome binding subunit of NAC can enter nuclei by several
routes.
|
Deletion of the first 27 amino acids of Egd1p abolishes Kap123p and
Pse1p dependent nuclear import
We examined the further deleted Egd1p-2GFP to define the domain that is
responsible for nuclear import by Kap123p and/or Pse1p. Nuclear accumulation
of N14-Egd1p-2GFP compared with
N11-Egd1p-2GFP was reduced and
the protein localized in the cytoplasm. Removal of 27 amino acids increased
this effect slightly (Fig. 6A).
Deletion up to 44 amino acids showed the same distribution as that seen in
N27-Egd1p-2GFP (data not shown). However, nuclear import was not
completely abolished. We always detected truncated Egd1p-2GFP in nuclei. The
remaining nuclear staining of
N14-Egd1p-2GFP and
N27-Egd1p-2GFP
was also observed in
kap123 and in the pse1-1 mutant
- even at nonpermissive temperature (Fig.
6B). Therefore, we assume that the region responsible for
Kap123p/Pse1p-dependent import is located between amino acids 11 and 27.
|
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DISCUSSION |
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Although the NAC subunits were small enough to enter the nucleus via
diffusion, we found evidence for active transport mechanisms. Our in vitro
transport assay revealed that full length Egd2p, a homologue of mammalian
NAC, as well as Egd1p, a BTF3 homologue, can be transported into the
nucleus by distinct importin
-proteins, the mammalian
1,
3,
7 and the yeast importin
Srp1p. The mammalian
importins
4 and
5 failed to import the yeast NAC proteins
although they carried other cargos such as nucleoplasmin in the same assay.
Competition of Egd2p-2GFP with nucleoplasmin confirmed the specificity of the
transport process. The weaker fluorescence signal found for import with yeast
importin
Srp1p in this assay had also been observed for other cargos
(Köhler et al.,
1999
).
The fact that the NAC proteins do not possess a classical NLS as described
previously (Makkerh et al.,
1996) does not contradict the
observed interaction with importin
proteins. Other proteins that lack
a classical NLS, such as RanBP3, are also known to be imported via the
importin
/importin ß-dependent pathway (Welch et al.,
1999
). We believe that the
result obtained for yeast NAC proteins in vitro is likely to reflect the in
vivo situation in mammals, since human
NAC protein can be
co-immunoprecipitated from HeLa-cell lysate using anti-importin
1/Rch1
antibodies (data not shown).
Our presumption of an active transport via the importin /importin
ß-dependent pathway was confirmed by in vivo experiments employing
temperature sensitive mutants of SRP1. It was shown that in the
srp1-31 mutant the nuclear import of classical NLS-bearing substrates
and of the ribosomal protein L11b was impaired (Loeb et al.,
1995
; Shulga et al.,
1996
; Stage-Zimmermann et al.,
2000
).
N11-Egd1p-2GFP
accumulated in the nucleus in wild-type cells but in srp1-31 cells
the truncated protein also localized in the cytoplasm at nonpermissive
temperature. In addition, we employed the srp1-49 mutant, which
exhibits defects in protein degradation and, to a lesser extent, in nuclear
transport (Tabb et al., 2000
)
and could not detect any significant difference compared with the wildtype. We
propose two explanations for the rather moderate effect of the SRP mutations.
First, a decrease in nuclear accumulation could not occur because transport
back to the cytosol was not possible. Only the import of newly synthesized
N11-Egd1p-2GFP was blocked. Second, there may exist alternative import
pathways. We tested this hypothesis by employing yeast nuclear import mutants
of ribosomal proteins, namely Kap123p and Pse1p.
Furthermore, we delineated the domain of Egd1p responsible for the
interaction with importins Kap123p/Pse1p. Whereas deletion of the first 11
amino acids abolished ribosome binding and led to a nuclear accumulation of
Egd1p, a further deletion of the next 3 amino acids drastically reduced the
Kap123p/Pse1p-dependent import of Egd1p. This region contains a KLXKL motif
that is found in all Egd1p homologues in the database. Additional deletion of
amino acids 15-27, a region that also bears a highly conserved cluster of
basic amino acids, enhanced the import defect only slightly. Both motifs are
not present in L25, the TATA-binding protein or Pho4, proteins known to be
imported by Pse1p/Kap123p (Schaap et al.,
1991; Kaffman et al.,
1998
; Pemberton et al.,
1999
). Our finding indicates a
close proximity or even partial overlap of the ribosome binding domain and one
of the nuclear localization signals in Egd1p. However, nuclear import was not
abolished completely when the first 27 amino acids were missing. Therefore, we
assume that Egd1p bears several nuclear import signals that trigger nuclear
transport by different pathways. One obvious candidate would be the Srp1p
(yeast importin
)-dependent pathway. This assumption could not be
tested because in the available temperature-sensitive mutants of
SRP1,
N14-Egd1p-2GFP is transported into nuclei efficiently at
permissive temperature. Thus, no difference in location of this protein was
detectable after the temperature shift before the cells were dead (data not
shown).
What is the reason for transport of NAC proteins into the nucleus? A
possible transcriptional function of yeast Egd1p as described by other authors
had been deduced from DNA gel shift experiments and moderately reduced amounts
of some mRNA species in an EGD1/BTT1-knockout strain (Parthun et al.,
1992; Hu and Ronne,
1994
). However, the
simultaneous deletion of all NAC subunits in yeast has no additional phenotype
(Reimann et al., 1999
) and
firm direct proof for a function of Egd1p as transcription factor is still
missing.
There are other processes that could demand a transient nuclear
localization of NAC subunits. Key components of translation, such as the
ribosomal subunits, 5S-RNPs and SRP assemble in the nucleus (Pederson and
Politz, 2000; Politz et al.,
2000
; Ciufo and Brown,
2000
). Pederson and Politz
propose nuclear assembly of these factors as an essential checkpoint during
the production of the translational apparatus. Also, other cytosolic factors
that are localized at steady state conditions in the cytosol and perform their
function in close conjunction with the ribosome, namely the SSB proteins, have
been demonstrated to reside transiently in the nucleus (Pfund et al.,
1998
; Shulga et al.,
1999
). Taking these facts into
consideration, another explanation of the NAC nuclear import could be that the
NAC proteins travel into the nucleus after their synthesis in the cytosol to
assemble with ribosomal subunits and are exported together with these
subunits. In contrast to the SSB proteins, NAC subunits are not exported by
the NES (nuclear export signal)-dependent factor Crm1p (J.F. et al.,
unpublished). Interestingly, efficient import of yeast NAC subunits requires
both Kap123p and Pse1p, similar to what has been found for ribosomal proteins.
We believe that the first efficient binding of NAC to ribosomal subunits
occurs in the nucleus. This interpretation would imply that yeast NAC does not
leave the ribosome when it releases the nascent polypeptide. The assumption is
supported by our observation that ER-bound ribosomes bear NAC and that binding
of in vitro translated yeast NAC to ribosomes is inefficient. One cannot
exclude the possibility that in the absence of sufficient ribosomal binding
sites the nuclear NAC is available for transcriptional processes. However, as
all our data are consistent with the idea of a co-regulated NAC assembly with
ribosomal subunits, we propose that the subunits of NAC enter the nucleus
primarily to bind to their final destination, the ribosome.
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ACKNOWLEDGMENTS |
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