From the
Control over the nuclear transport of transcription factors
(TFs) represents a level of gene regulation integral to cellular
processes such as differentiation, transformation and signal
transduction. The Saccharomyces cerevisiae TF SWI5 is excluded
from the nucleus in a cell cycle-dependent fashion, mediated by
phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear
entry occurs in G
Precisely scheduled nuclear import of transcription factors
(TFs)(
Proteins larger than
45 kDa require a nuclear localization sequence (NLS) (1) in
order to be targeted to the nucleus. Little is known, however,
concerning the regulation of import kinetics, which is likely to be
critical in cuing the aforementioned cell cycle transitions,
morphogenesis and transformation. In addition to the NLS, specific
signals carried by the transported proteins appear to function in a
regulatory fashion, whereby covalent modifications such as
phosphorylation may play a
role(3, 15, 16, 17, 18) .
We have demonstrated that the nuclear localization of hybrid proteins
in which fragments of the SV40 large T-antigen (T-Ag) are fused to the Escherichia coli
The yeast TF
SWI5 is involved in mating switch determination through regulating
transcription of the HO endonuclease(6) . It exhibits cell
cycle-dependent nuclear exclusion, entering the nucleus specifically in
G
We were interested in measuring
the kinetics of cdk site-dependent regulation of SWI5 nuclear transport
and decided to apply our previous approach of measuring the nuclear
uptake of bacterially expressed
E. coli expression plasmids for SWI5-
Figure 3:
Time course of nuclear import of SWI5
Figure 1:
The CDC28-k
sites within and C-terminal to the bipartite NLS of SWI5 inhibit
NLS-dependent nuclear transport of SWI5-
Figure 2:
Visualization of nuclear import of
SWI5-
The
cdk/CDC28 sites of the SWI5 CDN thus function to inhibit nuclear entry
of SWI5, determining the end point (Fn/c
We thank Reiner Peters (Institut für Medizinische
Physik, Münster, Germany) for support in preliminary studies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
.
-galactosidase fusion proteins
carrying SWI5 amino acids 633-682, including the nuclear
localization sequence (NLS:
Lys-Lys-Tyr-Glu-Asn-ValVal-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-ProArg-Lys
)
were analyzed for subcellular localization in appropriate
temperature-sensitive yeast strains blocked in G
or
G
/M using indirect immunofluorescence, and for nuclear
import kinetics in living rat hepatoma or Vero African green monkey
kidney cells microinjected with fluorescently labeled bacterially
expressed protein and quantitative confocal laser microscopy. Cell
cycle-dependent nuclear localization in yeast was both NLS and cdk
site-dependent, whereby mutation of the cdk site serines (Ser
and Ser
) to alanine resulted in constitutive
nuclear localization. In mammalian cells, the SWI5 fusion proteins were
similarly transported to the nucleus in an NLS-dependent fashion, while
the mutation to Ala of the cdk site serines increased the maximal level
of nuclear accumulation from about 1- to over 8-fold. We suggest that
phosphorylation at the cdk sites inhibits nuclear transport of SWI5,
consistent with our previous observations for the inhibition of SV40
large tumor antigen nuclear transport by phosphorylation by the cdk
cdc2. The results indicate for the first time that a yeast NLS and,
fascinatingly, its regulatory mechanisms are functional in higher
eukaryotes, implying the universal nature of regulatory signals for
protein transport to the nucleus.
)
is a key factor in the regulation of
gene expression and signal transduction. While proteins such as
histones appear to be constitutively targeted to the nucleus, others
are only translocated to the nucleus under specific conditions,
otherwise being cytoplasmic(1, 2, 3) . TFs
regulating nuclear gene expression are no different from other proteins
in terms of their being synthesized in the cytoplasm and thereby
subject to specific mechanisms regulating nuclear protein import. The
advantages of a conditionally cytoplasmic location for a TF include the
potential to control its activity by regulating its nuclear uptake, and
its direct accessibility to cytoplasmic signal-transducing systems. The
nuclear translocation of various TFs (2, 4, 5, 6, 7) and
oncogene products (8, 9, 10, 11) has
been shown to accompany changes in the differentiation or metabolic
state of eukaryotic cells precisely, indicating that nuclear protein
import is a key control point in the regulation of gene expression and
signal transduction. TFs able to undergo inducible nuclear import
include the glucocorticoid receptor(12) , the
-interferon-regulated factor ISGF-3(13) , the nuclear
v-jun oncogenic counterpart of the AP-1 transcription complex
member c-jun(14) , the Saccharomyces cerevisiae TF SWI5(6) , the Drosophila melanogaster morphogen dorsal(7) , and
NF-
B(2, 4, 5) .
-galactosidase enzyme is completely
dependent on the presence of the T-Ag NLS (amino acids
126-132)(15, 16) . However, the kinetics of
import are markedly enhanced by the presence of the N-terminal sequence
(amino acids 111-125) adjacent to the
NLS(15, 16) . The rate of nuclear import is regulated
by the casein kinase II (CKII) phosphorylation site
(Ser
)(16, 18) , while
phosphorylation at the cyclin-dependent kinase (cdk) cdc2 site
(Thr
) adjacent to the NLS determines the maximal extent
of nuclear accumulation(17) . The CKII and cdk sites, together
with the NLS, constitute the CcN motif, responsible for
phosphorylation-regulated T-Ag nuclear transport(17) . CKII
also appears to enhance nuclear import of the Xenopus laevis nuclear phosphoprotein nucleoplasmin(19) .
(6, 20) . This nuclear exclusion is
effected by phosphorylation by the cdk CDC28, the yeast equivalent of
cdc2(6, 20) , where three cdk sites, one of which is
within the spacer of the SWI5 bipartite NLS, are proposed to prevent
nuclear localization by inactivating NLS function through charge or
conformational effects (6) .
-galactosidase fusion proteins
microinjected into living mammalian cells using confocal laser
microscopy(16, 17, 18) . Fusion proteins
carrying SWI5 amino acids 633-682, including the NLS
(Lys-Lys-Tyr-Glu-Asn-Val-ValIle-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro-Arg-Lys
),
were found to be transported to the nucleus in an NLS-dependent
fashion, whereby the mutation to Ala of the CDC28 site serines
(Ser
and Ser
) in the vicinity of the SWI5
NLS increased the maximal level of nuclear accumulation from about 1-
to over 8-fold. These results were consistent with qualitative findings
for subcellular localization of the same SWI5-galactosidase fusion
proteins in appropriate yeast strains. The results indicate that a
yeast NLS together with its regulatory mechanisms are functional in
higher eukaryotes, implying the universal nature of constitutive and
regulatory signals for protein transport to the nucleus. A cell
cycle-dependent NLS (``CDN''), able to function in diverse
eukaryotic cell types, is thus functionally defined here for the first
time.
Chemicals and
Reagents
Isopropyl-1-thio--Dgalactopyranoside,
-galactosidase (EC 3.2.1.23.37), and polyethylene glycol 1500 were
from Boehringer Mannheim, and 5-iodacetamidofluorescein from Molecular
Probes. Other reagents were from the sources described
previously(16, 17, 18) .
Yeast Strains
The S. cerevisiae strains
used were ``wild type'' K1258 (Mata/Mata, can1-100, ura3, ho) and the
temperature-sensitive cell cycle mutants K1509 (Mat, cdc13-1, ura3, ho) and K1550
(Mata, cdc28-4, can1-100, ura3, ho)(6, 21) .
Cell Culture
HTC rat hepatoma tissue culture (a
derivative of Morris hepatoma 7288C) and Vero (African green monkey
kidney) cell lines were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf
serum(16, 17, 18) .
SWI5/
To prepare the SWI5--Galactosidase Fusion
Proteins
-galactosidase fusion
proteins, PCR was used to generate a fragment of SWI5 encoding amino
acids residues Ser
to Leu
. The
oligonucleotides employed were used to design an initiation
(methionine) codon immediately 5` of the SWI5 sequence and to produce
an EcoRI-HindIII fragment, the sequence of which was
verified by DNA sequencing. The fragment was used to replace the EcoRI-HindIII fragment of YCpGAL/SWI5 (22) and the E. coli
-galactosidase gene was
inserted as a HindIII fragment from plasmid
pMC1871(23) . The SWI5 (633-682: CDC28
)
and SWI5 (633-682: NLS
)
-galactosidase fusion proteins
are expressed from the GAL1-10 promoter.
-galactosidase fusion proteins,
exactly comparable to those for expression in yeast, were derived by
inserting the SWI5 sequences as EcoRI
(blunt-ended)-HindIII fragments into the polylinker of the
T7-expression vector pMWT7 restricted with NdeI
(blunt-ended)/HindIII. The lacZ gene from plasmid
pMC1871 was inserted C-terminal to the SWI5 sequences as a HindIII fragment. E. coli expression plasmids for
fusion proteins in which SV40 T-Ag amino acids 111-135 are fused
to the N terminus of E. coli
-galactosidase (amino acids
9-1023) have been described(15, 16) .
Immunofluorescence
SWI5--galactosidase fusion
proteins were localized in fixed yeast cells as described
previously(6, 21) using mouse monoclonal
anti-
-galactosidase antibody, followed by a rhodamine-conjugated
goat anti-mouse secondary antibody. Nuclei were visualized by mounting
the cells in the DNA-specific dye diamidinophenyl indole (DAPI).
Fusion Protein Expression
1 mM
isopropyl-1-thio--Dgalactopyranoside was used in media to
induce fusion protein expression. Proteins were purified by affinity
chromatography and labeled with 5-iodacetamidofluorescein as
described(15) .
Microinjection and Laser Microscopy
HTC cells were
fused with polyethylene glycol about 1 h prior to microinjection to
produce polykaryons(15) . Microinjection and single-cell
microscopic fluorescence measurements on HTC and Vero cells were
performed as
previously(15, 16, 17, 18) .
Quantitation of fluorescence using confocal microscopy and
microphotolysis has been described previously in detail (see (24, 25, 26, 27) for applications).
Histochemical staining of fixed cells for -galactosidase activity
after microinjection was performed as described(15) .
The Cell Cycle-dependent NLS of SWI5 Can Target a
Heterologous Protein to the Nucleus in Yeast in an NLS- and cdk
Site-dependent Fashion
The bipartite NLS of SWI5 residing within
amino acids 633 and 682 (which also includes the cdk/CDC28 sites at
Ser and Ser
) has been shown previously to
be sufficient and necessary to confer cell cycle-dependent nuclear
localization on both SWI5 itself and the heterologous E. coli protein
-galactosidase in yeast(6) . We were
interested in investigating the sequences responsible for this cell
cycle-dependent regulation, as well as examining the nuclear import
kinetics. We made SWI5-
-galactosidase fusion protein derivatives
(amino acid sequences are shown in Fig. 3) for expression both
in yeast under the control of the GAL1-10 promoter, and in
bacteria, to enable the examination of nuclear import of microinjected
protein in mammalian cells.
-galactosidase fusion proteins in vivo in microinjected
HTC cells. Results represent the average of at least 8 single-cell
measurements (S.D. not greater than 28% the value of the mean) for Fn/c
(fluorescence quantitated in the nucleus, relative to that in the
cytoplasm, as determined by fluorescence microphotolysis; see
``Materials and
Methods'')(16, 17, 18) . The results for
nuclear transport rates determined by curve-fitting (17) are
shown in Table 1(see legend).
SWI5 amino acids 633-682 are
capable of conferring cell cycle-dependent nuclear localization on the
heterologous protein -galactosidase, the protein being nuclear in
G
(Fig. 1A) and cytoplasmic during the rest
of the cell cycle (e.g. in G
; see Fig. 1B)(6) . This nuclear localization was
dependent on the SWI5 bipartite NLS (amino acids 636-655) (6) since the SWI5-
NLS construct exhibited strictly
cytoplasmic localization, even in cells arrested in G
(Fig. 1A). When the cdk/CDC28 site serines 646
and 664 were replaced by alanine residues, the SWI5-
-galactosidase
protein was constitutively nuclear in contrast to wild type, even in
G
(Fig. 1B). The CDN of SWI5 could thus be
defined as the bipartite NLS together with the cdk sites
(Ser
/Ser
), able to confer cell
cycle-dependent nuclear localization on a heterologous protein.
-galactosidase fusion
proteins. A, cdc28-4 cells expressing the SWI5
-galactosidase constructs as indicated were arrested in G
by incubation at the restrictive temperature (36 °C) and the
distribution of fusion proteins revealed by staining with a mouse
monoclonal anti-
-galactosidase antibody, followed by a
rhodamine-conjugated goat anti-mouse secondary antibody. The leftpanels show rhodamine fluorescence, and the rightpanels the same fields stained with DAPI. B, cdc13-1 cells expressing the SWI5
-galactosidase constructs indicated were treated as in A above to effect arrest in G
/mitosis. Left-hand
panels show rhodamine fluorescence, and right-handpanels DAPI fluorescence.
The CDN of SWI5 Confers NLS- and cdk Site-dependent
Nuclear Transport in Mammalian Cells
In vivo nuclear
import kinetics of the fluorescently labeled SWI5 -galactosidase
fusion proteins were measured after microinjection in HTC polykaryons
or Vero cells ( Fig. 2and 3, Table 1). Results for both
cell lines were completely comparable (Fig. 2, and not shown) in
that the wild type SWI5 construct was only weakly transported to the
nucleus in interphase cells, in contrast to the CDC28
site-mutated derivative, which localized in the nucleus very
strongly and rapidly. This nuclear localization was clearly
NLS-dependent, since the SWI5-
NLS derivative was exclusively
cytoplasmic even 2 h after microinjection (Fig. 2).
-galactosidase fusion proteins in vivo in
microinjected HTC polykaryons (A) and Vero (B) cells.
In the case of panel A, fluorescent visualization is shown 15 (D) and 30 (A and G) min after
microinjection. Other panels show histochemical staining for
-galactosidase activity, where cells were fixed and stained as
described under ``Materials and Methods'' (15) 15 (E), 30 (B and H) and 120 (C, F, and I) min, respectively, after microinjection. In
the case of panel B, fluorescent visualization is
shown 10 min (A), and staining for
-galactosidase
activity 15 (B), 30 (C), and 120 (D) min
after microinjection, respectively.
Quantitative results (Fig. 3, Table 1) for the
SWI5--galactosidase fusion proteins confirmed these observations,
whereby the CDC28
site-mutated derivative was
accumulated to levels about 8 times that in the cytoplasm within about
40 min after microinjection. As shown in Table 1, these transport
properties are comparable to those for T-Ag-
-galactosidase fusion
proteins containing the T-Ag CcN motif (the NLS together with the
requisite phosphorylation
sites)(15, 16, 17, 18) . Similar
results were obtained using the same SWI5 fusion proteins in our
mechanically perforated HTC cell in vitro nuclear transport
system (see (17) ) (not shown). Nuclear transport kinetics were
examined mostly in interphase cells principally for reasons of
convenience with respect to both microinjection and HTC cell
perforation, but the CDC28
SWI5 fusion protein
derivative appeared to be transported constitutively to the nucleus,
irrespective of the stage of the cell cycle (not shown).
) of nuclear import (see Table 1). This is consistent with our observations concerning
cdk/cdc2 site-mediated inhibition of nuclear transport of
T-Ag(17) , where phosphorylation adjacent to the T-Ag NLS
reduces the maximal extent of nuclear accumulation of T-Ag fusion
proteins. The mechanisms of the cdk site-mediated effects appear to be
different; whereas cdc2 inhibition of T-Ag transport appears to be
through phosphorylation increasing the affinity of T-Ag for a putative
cytoplasmic retention factor(17) , the CDC28 sites in SWI5
appear to inhibit nuclear localization by inactivating or masking the
function of the NLS (see (6) ). NLS masking by phosphorylation
has been reported to regulate nuclear transport of both lamin B2
(inhibited by phosphorylation at two protein kinase C sites
N-terminally adjacent to the NLS)(28) , and the actin-binding
protein cofilin (whose nuclear translocation upon heat shock is
accompanied by dephosphorylation at a multifunctional
calmodulin-dependent protein kinase site adjacent to the putative
NLS)(29, 30) .
CDN/cdk-mediated Regulation of Nuclear Protein
Import
A number of examples of cell cycle-dependent
phosphorylation (cdk)-mediated regulation of nuclear entry are known,
the best examples being those of T-Ag(17) , SWI5 ( (6) and see above), and the ``retinoblastoma
susceptibility factor'' tumor-suppressor gene p110,
whose tightness of association with the nucleus (``nuclear
tethering'') appears to be reduced by cell cycle-dependent
phosphorylation(31, 32) . Table 2lists proteins
whose nuclear localization is specifically regulated during the cell
cycle or known to be regulated by cdks and/or CDNs. Although their
respective activities themselves may be cdk-regulated, kinases other
than cdks (e.g. CKII in the case of the Drosophila lodestar protein; see Table 2) (34) also appear to
be capable of regulating cell cycle-dependent nuclear entry. CDNs may
well be a general mechanism of controlling protein import with respect
to the cell cycle, enabling precise timing of nuclear entry as
required.
In conclusion, this study shows that the SWI5 CDN can
confer NLS- and cdk site-dependent nuclear transport on a heterologous
protein in mammalian cells. Importantly, the results show that a
bipartite yeast NLS and, fascinatingly, its regulatory mechanisms are
functional in higher eukaryotes, implying the universal nature of
constitutive and regulatory signals for protein transport to the
nucleus (see Refs. 3, 41, and 42). The regulation of nuclear protein
import through cell cycle-dependent or other phosphorylation may be as
universal as NLSs themselves(3, 17) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.