From the Department of Physiology, McGill University, Montreal, Province of Quebec H3G 1Y6, Canada
Received for publication, January 16, 2001, and in revised form, March 6, 2001
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ABSTRACT |
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Nuclear import of proteins that are too large to
passively enter the nucleus requires soluble factors, energy, and a
nuclear localization signal (NLS). Nuclear protein transport can be
regulated, and different forms of stress affect nucleocytoplasmic
trafficking. As such, import of proteins containing a classical NLS is
inhibited in starving yeast cells. In contrast, the hsp70 Ssa4p
concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p
in starving cells is reversible, and transfer of stationary phase cells
to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. In
starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear
accumulation, and a short hydrophobic sequence, termed Star (for
starvation), is sufficient to localize the reporter proteins green
fluorescent protein or In eukaryotic cells, DNA replication and RNA synthesis take place
in the nucleus, whereas protein synthesis occurs in the cytoplasm.
Proper communication between these processes depends on the transport
of soluble factors between both compartments. Nucleocytoplasmic
trafficking requires that proteins cross the nuclear envelope. To do
so, proteins travel through nuclear pore complexes, large specialized
structures that span both the inner and outer nuclear membrane.
Proteins that are smaller than 40-60 kDa can diffuse through nuclear
pore complexes without the requirement of energy. In contrast,
macromolecules with a molecular mass larger than 40-60 kDa
enter the nucleus via active transport (reviewed in Refs. 1 and 2).
A variety of pathways mediate the nuclear accumulation of proteins too
large to diffuse into the nucleus (reviewed in Refs. 1 and 2). In most
cases, nuclear trafficking depends on specific carrier molecules termed
importins. In the yeast Saccharomyces cerevisiae 14 members
of the In addition to the well characterized classical nuclear import route, a
variety of nonclassical pathways have been described that depend on
other members of the During logarithmic growth, cells deplete their medium for nutrients and
enter stationary phase. Under these conditions, a variety of genes
modify their expression levels. Nutrient depletion represents a
specific form of stress that inhibits classical nuclear transport and
can also affect nonclassical transport pathways (7). Moreover, various
proteins regulating the response to changing nutrient concentrations,
such as Mig1p, may alter their distribution between nucleus and
cytoplasm (reviewed in Ref. 8).
Different forms of stress alter the cellular physiology and may damage
cells. To survive and recover from stress-induced injury, cells need to
activate specialized survival and repair mechanisms. In particular,
heat shock proteins of the hsp70/hsc70 family play an essential role in
these processes. Hsp70/hsc70s shuttle between nucleus and cytoplasm (9)
and accumulate in nuclei upon exposure to heat stress. Unlike heat
shock, however, the effect of starvation on hsp70 localization has not
been studied in detail.
The yeast S. cerevisiae contains six members of the
cytosolic hsp70 family. Cytosolic hsp70s are divided into two
subfamilies: four SSA-encoded gene products (Ssa1p to Ssa4p)
and two SSB gene products (Ssb1p and Ssb2p) (reviewed in
Ref. 10). The SSA4 gene is particularly interesting as its
expression is highly induced upon stress (10) and up-regulated during
diauxic shift. Proteins of the SSA family are located
in the cytoplasm and the nucleus, because they shuttle between both
compartments under normal conditions. Unlike gene products of the
SSA group, Ssb1p and Ssb2p are located predominantly in the
cytoplasm. Ssb1p is prevented from nuclear accumulation due to the
presence of a nuclear export signal in its N-terminal domain (11).
It has not been analyzed previously whether nutrient depletion affects
the localization of hsp70s. We have addressed this problem by
generating fusions containing Ssa4p and GFP (Aequorea victoria green fluorescent protein). We now show that upon
starvation GFP fusion proteins carrying Ssa4p or its N-terminal domain
accumulate in nuclei. We have identified a short hydrophobic segment in
this N-terminal portion, referred to as the Star sequence, which is sufficient to direct non-nuclear reporter proteins to nuclei when cells
are entering stationary phase. Moreover, we demonstrate that the
Yeast Strains and Growth of Cells--
Yeast strain RS453
(ade2 ADE3 leu2 ura3 trp1 his3), provided by V. Doye
(Paris), was used as wild type strain. Mutant yeast strains were
provided by G. Schlenstedt (Hamburg, Germany). Upon transformation,
cells were grown in synthetic complete medium lacking uracil or leucine
(dropout medium), depending on the selectable marker introduced by the
plasmid. Cells were kept at room temperature unless indicated
otherwise. Expression of genes controlled by the GAL1 or
GALS promoter was induced with 2% galactose as a carbon source. For data shown in Fig. 2, cells were transferred for 6 h
into fresh glucose-containing medium. The following inhibitors were
added as indicated in the Fig. 2: 2 mM hydrogen peroxide (American Chemicals, Montreal), 2 mM diethyl maleate
(Sigma), or 100 µg/ml cycloheximide (Sigma).
Plasmids and Transformation of Yeast Cells--
All plasmid
constructions were carried out in Escherichia coli strain
XL1-Blue. The correctness of constructs was verified by sequencing with
the Sequenase 2.0 kit (U. S. Biochemical Corp.). To generate fusions
between GFP and SSA4, the GFPmut1 allele was used. GFPmut1
encodes a protein with ~35 times the fluorescence intensity of the
wild type (12). Plasmid pGAD-GFP encodes NLS-GFP, a fusion protein that
carries the classical SV40-NLS (13). pGAD-GFP was kindly provided by
Dr. D. Goldfarb (Rochester, NY). For GFP-Ssa4p-(16-642), a
NotI-linker (12-mer, New England Biolabs, Mississauga, ON)
was inserted into the unique NlaIII site of SSA4,
thereby generating plasmid p930. Plasmid p930 was cut with
NotI, and the GFP-coding sequence was fused in-frame to
codon 16 of SSA4; the fusion gene encodes
GFP-Ssa4p-(16-642). For expression in yeast, the centromeric plasmid,
which carries the URA3 selectable marker, was used as a
vector. Expression of the gene fusions inserted into this plasmid is
regulated by the GAL1 promoter. For generation of GFP-Star, GFP containing a NotI linker at the 3'-end (14) was fused to oligonucleotides encoding amino acid residues Ile-162 to Ile-171 of
Ssa4p, followed by an UAG stop codon. To generate fusions between the
Star sequence and Fluorescence Microscopy--
GFP-containing reporter proteins
were located by fluorescence microscopy as previously described (14).
Fusion proteins carrying GFP-Ssa4p Fusion Proteins Accumulate in Nuclei of Starving
Cells--
Fusions of GFP and members of the SSA family of
proteins are proper models to study their targeting within the cell
(11). As described below, we have used proteins containing GFP and
distinct segments of the cytoplasmic hsp70 Ssa4p for our studies. In
logarithmically growing cells, the fusion protein GFP-Ssa4p-(16-642),
which contains amino acid residues 16-642 of the authentic sequence of
Ssa4p (642 amino acid residues), was both nuclear and cytoplasmic (Fig. 1, A and B). By
contrast, this protein accumulated in nuclei of stationary phase cells
(Fig. 1, C and D). GFP-Ssa4p-(16-642) could exit
the nucleus when cells were provided with fresh medium containing glucose (Fig. 1, E and F). This appearance in the
cytoplasm did not reflect de novo gene expression, since
glucose inhibits transcription from the GAL1 promoter.
Export of GFP-Ssa4p-(16-642) from the nucleus was independent of the
carbon source in the growth medium as the same result was obtained for
cells incubated with nonfermentable substrates such as glycerol (Fig.
1, G and H). Furthermore, nuclear export of
GFP-Ssa4p-(16-642) was an active process that was inhibited if cells
were kept at 4 °C (Fig. 1, I and J). For
comparison, we have analyzed the localization of the GFP tag and
NLS-GFP. GFP distributed throughout the nucleus and cytoplasm under
different growth conditions (Fig. 1, K-N). NLS-GFP, a
substrate for classical nuclear transport, concentrated in nuclei of
control cells but equilibrated between nucleus and cytoplasm in
starving cells (Fig. 1, O-R, and Ref. 7).
Nuclear Export of GFP-Ssa4p-(16-642) Is Sensitive to Oxidative
Stress--
Nuclear accumulation of GFP-Ssa4p-(16-642) in starving
cells was reversible, and transfer to fresh medium promoted its export into the cytoplasm. Nucleocytoplasmic trafficking and, in particular, nuclear export of several proteins is sensitive to oxidants (Refs. 16
and 17; reviewed in Ref. 8). When stationary phase cells were
transferred to fresh medium containing glucose, both hydrogen peroxide
and diethyl maleate strongly inhibited nuclear export of the fusion
protein (Fig. 2, C-F). In
contrast, the protein synthesis inhibitor cycloheximide had no effect
on nuclear export, and GFP-Ssa4p-(16-642) appeared in the cytoplasm,
as was observed in control cells (compare A and B
with G and H in Fig. 2).
The N-terminal Domain of Ssa4p Targets GFP to Nuclei of Starving
Cells--
To define regions of Ssa4p that are sufficient for nuclear
accumulation when cells enter stationary phase, a fusion of the authentic 236 amino acid residues of Ssa4p to GFP was generated. This
fusion protein, referred to as Ssa4p-(1-236)-GFP, lacks the bipartite
NLS present in Ssa4p. In logarithmically growing cells, Ssa4p-(1-236)-GFP was found in the cytoplasm and the nucleus (Fig. 3, A and B),
similar to GFP-Ssa4p-(16-642). Moreover, Ssa4p-(1-236)-GFP concentrated in nuclei of starving cells (Fig. 3, C and
D), demonstrating that the N-terminal segment of Ssa4p was
sufficient to promote nuclear accumulation in stationary phase cells.
As a control for a non-nuclear protein, the distribution of
GFP- A Short Hydrophobic Segment of the N-terminal Ssa4p Domain Is
Sufficient for Nuclear Accumulation in Starving Cells--
Amino acid
residues 162-171 of Ssa4p are mostly hydrophobic. The sequence
Ile-Ala-Gly-Leu-Asn-Val-Leu-Arg-Ile-Ile (referred to as Star sequence,
for starvation) fits the consensus sequence proposed for hydrophobic
nuclear export signals (16). However, when fused to GFP, we did not
detect nuclear exclusion (data not shown), but GFP-Star equilibrated
between nucleus and cytoplasm in exponentially growing cells (Fig. 3,
E and F). Importantly, GFP-Star concentrated in
nuclei of starving cells (Fig. 3, G and H).
Three different mechanisms could explain nuclear accumulation of
GFP-Star in nutrient-depleted cells. In one scenario, due to its small
size, GFP-Star diffuses into nuclei and may be retained when cells are
starving. This would involve an interaction with nuclear proteins which
provide anchors that prevent GFP-Star from diffusing into the
cytoplasm. Alternatively, the Star sequence could function as an NLS
that is active only under certain physiological conditions. Finally, a
combination of nuclear retention and nuclear import may concentrate in
nuclei reporter proteins containing the Star sequence. To begin to
address these questions, we have generated the fusion protein
Star- GFP-Star Accumulates in Nuclei of Yeast Strains Carrying Mutations
in Nmd5p Is Required to Accumulate Star-
As a possible complication of our experiments, overexpression of
Star- Changes in physiological conditions require cells to adapt and, in
response to various types of stress, proteins may alter their
intracellular localization. In particular, trafficking between nucleus
and cytoplasm is affected in cells exposed to oxidative or osmotic
stress (reviewed in Ref. 8). Furthermore, changes in nutrient
concentration have been shown to alter protein localization (6, 19,
20). For instance, the distribution of regulatory and catalytic
subunits of protein kinase A in S. cerevisiae depends on the
carbon source (20). Likewise, the distribution of hepatic glucokinase
is altered in response to metabolic changes (19). The effect of
nutrient depletion on nucleocytoplasmic transport of heat shock
proteins, however, has not been defined previously.
Proteins of the hsp70 family are key components in the stress response,
a process that is conserved among eukaryotes. We have now analyzed the
effect of starvation on Ssa4p localization in budding yeast. To this
end, distinct segments of Ssa4p were fused to the non-nuclear reporter
proteins GFP or Proteins that exceed the diffusion radius of the nuclear pore complex,
like Star- NMD5 is a nonessential gene that encodes a nuclear carrier
which imports the mitogen-activated protein kinase Hog1p and the transcription elongation factor TFIIS into nuclei (5, 21). We have now
identified the Star sequence as a signal that mediates nuclear
transport via Nmd5p. Nuclear import via Nmd5p could occur by direct
binding of a Star-containing protein to Nmd5p or by a piggyback
mechanism that involves a linker molecule between Nmd5p and the Star
sequence. At present, targeting signals recognized by Nmd5p are not
defined, and future experiments will have to determine whether Nmd5p
and the Star sequence interact directly. It should be noted that we did
not detect nuclear exclusion of Star- Like Star--galactosidase to nuclei. To determine
whether nuclear accumulation of Star-
-galactosidase depends on a
specific nuclear carrier, we have analyzed its distribution in mutant
yeast strains that carry a deletion of a single
-importin gene. With
this assay we have identified Nmd5p as a
-importin required to
concentrate Star-
-galactosidase in nuclei when cells enter
stationary phase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-importin family have been identified (reviewed in Ref. 3),
three of which are essential for cell viability. These carriers are
involved in nuclear export or import of proteins and RNA. At present,
classical nuclear protein transport is the pathway understood best.
This transport route requires an adaptor protein,
-importin (Srp1p
in yeast) that links the nuclear cargo to
-importin (Rsl1p in
yeast). Like other forms of nuclear transport, classical nuclear
protein import requires energy, soluble transport factors, and nuclear
localization sequences (NLSs),1 specialized signals
that target proteins into the nucleus. In classical nuclear transport,
these signals can be of two types: monopartite or bipartite.
Monopartite signals are simple stretches of basic amino acids, whereas
bipartite NLSs contain two clusters of basic amino acids separated by a
spacer region (reviewed in Ref. 4).
-importin family of carriers (3). Frequently,
nonclassical transport pathways are mediated by NLSs distinct from the
simple or bipartite type, and such nonclassical NLSs do not share a
consensus sequence. Examples of proteins containing a nonclassical NLS
include the mammalian protein hnRNPA1 and the yeast protein Npl3p (1,
2). In general, proteins bearing a nonclassical NLS associate directly
with a specific
-importin for targeting to the nucleus, without the
requirement of an adaptor. Thus,
-importins have been shown to play
a role in nuclear import of ribosomal proteins, transcription factors,
or RNA-binding proteins (3). Of particular interest are
-importins
involved in trafficking of proteins that regulate the response to
stress or changes in nutrient availability. For instance, Nmd5p
mediates nuclear import of the mitogen-activated protein kinase Hog1p
upon exposure to osmotic stress, and nuclear import is triggered by
Hog1p phosphorylation (5). Another example of regulated
nucleocytoplasmic trafficking is Mig1p, a transcription factor
promoting glucose-mediated repression of several genes (6). Removal of
glucose from the growth medium induces Mig1p phosphorylation via Snf1p.
Phosphorylated Mig1p is then recognized by the
-importin Msn5p and
subsequently exported to the cytoplasm (6).
-importin Nmd5p is required to import a fusion between the Star
sequence and
-galactosidase into nuclei of early stationary phase cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase, oligonucleotides encoding methionine followed by the Star sequence were fused to the 5'-end of
the
-galactosidase gene. To obtain GFP-
-galactosidase, enhanced green fluorescent protein (CLONTECH, Palo Alto, CA)
was fused in-frame to the 5'-end of
-galactosidase. For expression
in yeast, the Star-
-galactosidase gene was cloned into centromeric
plasmids with either LEU2 or URA3 as a selectable
marker. Yeast cells were transformed as described previously
(14) and selected on dropout plates containing 2% glucose.
-galactosidase were located with monoclonal
antibodies as detailed in Ref. 15. To detect GFP-Star and
Star-
-galactosidase simultaneously, cells were grown in synthetic
complete medium lacking both uracil and leucine and containing 2%
galactose. Cells were fixed, permeabilized, and incubated overnight
with monoclonal antibodies against
-galactosidase (diluted 1:500)
and polyclonal antibodies against GFP (1:50,
CLONTECH). Primary antibodies were detected with
Cy3-conjugated affinity-purified antibodies against mouse IgG and
fluorescein isothiocyanate-conjugated affinity-purified antibodies to
rabbit IgG (Jackson ImmunoResearch, West Grove, PA). DNA was visualized
with 4',6-diamidino-2-phenylindole (DAPI), and slides were mounted in
Vectashield (Vector Laboratories, Burlingame, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Localization of GFP-Ssa4p-(16-642),
GFP, and NLS-GFP in starving cells. Yeast cells synthesizing
GFP-Ssa4p-(16-642) (A-J), GFP (K-N), or
NLS-GFP (O-R) were grown in medium containing galactose at
30 °C either overnight (control, A, B,
K, L, O, P) or for 2 days
(stationary, C, D, M, N,
Q, R). Stationary phase cells were transferred to
fresh medium supplemented with glucose (E, F,
I, J) or glycerol (panels G and
H). After 6 h of growth in glucose-containing medium
(E and F) or overnight growth on glycerol
(panels G and H), GFP-Ssa4p(16-642) was
localized. For comparison, cells were kept overnight in medium with
glucose at 4 °C (I and J). DAPI-staining of
DNA (A, C, E, G,
I, K, M, O, Q)
and green fluorescence of reporter proteins (B,
D, F, H, J, L,
N, P, R) are shown.
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Fig. 2.
Nuclear export of GFP-Ssa4p-(16-642) is
sensitive to oxidative stress. Stationary phase cells were
transferred to fresh glucose-containing medium in the presence 2 mM hydrogen peroxide (C and D), 2 mM diethyl maleate (DEM) (E and
F), or 100 µg/ml cycloheximide (chx)
(G and H). Controls were transferred to fresh
medium without any addition (A and B). DNA was
stained with DAPI (A, C, E,
G), and fusion proteins were located by green fluorescence
(B, D, F, H).
-galactosidase was also monitored. This protein was cytoplasmic,
with nuclei and vacuoles being excluded (Fig. 3, I and
J). It should be noted that nuclear accumulation of
GFP-Ssa4p-(16-642), Ssa4p-(1-236)-GFP, GFP-Star, or
Star-
-galactosidase (see below) was never complete, and a
portion of the protein was always detected in the cytoplasm. Nuclear
accumulation was also transient and preferentially detected in early
stationary phase cells.
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Fig. 3.
Ssa4p-(1-236)-GFP and GFP-Star
accumulate in nuclei of starving cells. Ssa4p-(1-236)-GFP,
GFP-Ssa4p-(162-171), termed GFP-Star, and GFP- -galactosidase were
localized in exponentially growing cells (A, B,
E, F, I, J) and in
stationary phase cells (C, D, G,
H). DNA (A, C, E,
G, I), Ssa4p-(1-236)-GFP (B and
D), GFP-Star (F and H), and
GFP-
-galactosidase (J) were located by fluorescence
microscopy (A-I) or indirect immunofluorescence
(J).
-galactosidase. This fusion protein is too large to translocate
across the nuclear envelope by passive diffusion; therefore, its
nuclear localization would require transport into nuclei by an active
process. As shown in Fig. 4,
Star-
-galactosidase was able to accumulate in nuclei of stationary
phase cells, supporting the idea that the Star sequence can function as
an NLS. To compare the distribution of GFP-Star and
Star-
-galactosidase under identical conditions, both proteins were
synthesized in the same cell and localized in logarithmically growing
as well as stationary phase cells (Fig. 4, A-F). GFP-Star and Star-
-galactosidase colocalized and concentrated in nuclei when
cells were entering stationary phase (Fig. 4, D-F). Upon prolonged incubation of cells for several days in nutrient-depleted medium, the levels of GFP-Star and Star-
-galactosidase were reduced, and we no longer detected a concentration of the reporter proteins in
nuclei (data not shown). This result most likely reflects protein degradation in starving cells.
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Fig. 4.
Localization of GFP-Star and
Star- -galactosidase. Exponentially
growing (A, B, C) and starving yeast
cells (D, E, F) synthesizing GFP-Star
and Star-
-galactosidase (Star-
-gal) were located by
indirect immunofluorescence. Staining of the DNA (A and
D), GFP-Star (B and E), and
Star-
-galactosidase (C and F) are shown.
-Importin Genes--
We have tested the nuclear accumulation of
GFP-Star in yeast strains carrying a deletion
in one of the
-importin genes. With the experiments shown in Fig. 5 and summarized in Table
I, we followed the localization of
GFP-Star in cells during logarithmic growth and in stationary phase.
All of the mutants tested equilibrated GFP-Star between nucleus and
cytoplasm when cells were growing logarithmically. However, GFP-Star
accumulated in nuclei when cultures were in early
stationary phase. These results are consistent with the interpretation
that the concentration of the small protein GFP-Star in nuclei does not
depend on one particular
-importin and may be achieved by nuclear
retention only.
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Fig. 5.
Nuclear accumulation of the small protein
GFP-Star in -importin mutants. Yeast
cells carrying a deletion of the
-importin genes YRB4
(A-D), KAP114 (E and F)
and NMD5 (G and H) were monitored for
the distribution of GFP-Star in logarithmic phase (B) or
early stationary phase (D, F, H). For
comparison, nuclei were stained with DAPI (A, C,
E, G).
Distribution of GFP-Star and Star--galactosidase in wild type and
mutant cells
-importin genes, GFP-Star and Star-
-galactosidase were localized.
Cells were analyzed during logarithmic growth and when entering
stationary phase, as indicated. It is shown whether the signal was
detected in nuclei (N) or cytoplasm (C). N + C denotes that
reporter proteins were detected in nucleus and cytoplasm; nuclear
accumulation was seen for N>C. Alternative names of the
-importin
genes that have been inactivated are given in parentheses.
-galactosidase in Nuclei of
Early Stationary Phase Cells--
To determine whether the deletion of
a
-importin gene affects active nuclear import mediated by the Star
sequence, we monitored the distribution of Star-
-galactosidase
during logarithmic and stationary phases of the culture. As shown in
Fig. 6 and summarized in Table I, most of
the deletion mutants were able to accumulate Star-
-galactosidase in
nuclei, supporting the idea that they are not required for nuclear
import mediated by the Star sequence. However, cells lacking a
functional NMD5 gene failed to concentrate Star-
-galactosidase in nuclei of early stationary phase cells (Fig.
6, Table I), indicating that Nmd5p plays a role in these transport
reactions.
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Fig. 6.
Distribution of
Star- -galactosidase in
-importin mutants. Star-
-galactosidase
(Star-
-gal) was localized in early stationary
phase cells in mutant yeast strains lacking functional genes for
YRB4 (A and B), KAP114
(C and D), or NMD5 (E and
F). Star-
-galactosidase was detected with monoclonal
antibodies against
-galactosidase (B, D,
F), and nuclei were visualized with DAPI (A,
C, E).
-galactosidase may result in high levels of cytoplasmic protein, which could mask its nuclear accumulation. To obtain lower
levels of gene expression, we cloned the Star-
-galactosidase gene
into vectors carrying the GALS promoter (18). However, when
we localized Star-
-galactosidase in
nmd5::TRP1 cells transformed with these
plasmids, the reporter protein did not concentrate in nuclei when cells
entered stationary phase (data not shown). These results further
support the idea that Nmd5p is involved in nuclear import mediated by
the Star sequence.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase. Both GFP-Ssa4p-(16-642) and
Ssa4p-(1-236)-GFP concentrated in nuclei of starving cells during
early stationary phase, indicating that the N-terminal domain of Ssa4p
is sufficient for nuclear accumulation. Because Ssa4p-(1-236)-GFP is
lacking the bipartite NLS present in authentic Ssa4p, a classical NLS
is not required to concentrate the protein in the nuclei of starving
cells. Within the N-terminal domain of Ssa4p we have identified a short
stretch of 10 mostly hydrophobic amino acid residues, termed Star (for
starvation), which is sufficient to promote nuclear concentration in
nutrient-depleted cells. Thus, both GFP-Star as well as
Star-
-galactosidase accumulate in nuclei of early stationary phase
cells. Because the sequence context of Star is different in both fusion
proteins, it rules out the possibility that an artificial nuclear
targeting sequence was generated by linking the Star sequence to a
reporter protein.
-galactosidase, must enter the nucleus by active
transport, whereas small molecules can diffuse across nuclear pore
complexes. We have demonstrated that Star-
-galactosidase concentrates in nuclei of starving cells, whereas NLS-GFP, a substrate for classical nuclear protein import, failed to accumulate. This suggests differences in the requirements for nuclear accumulation and
the cellular apparatus mediating Star-
-galactosidase and classical
NLS-dependent import. Our hypothesis is further supported by analyses of mutant yeast strains that carry a deletion of one of the
-importin genes, since we have identified Nmd5p as a
-importin that plays an essential role in nuclear accumulation of
Star-
-galactosidase when cells enter stationary phase. By contrast,
NLS-GFP is believed to be imported into nuclei by the classical nuclear
import pathway, requiring
-importin and the
-importin Kap95p, and
this pathway is inactivated in starving cells. Moreover, nuclear
accumulation in starving cells is not a general feature of proteins
carrying a nonclassical NLS. For instance, the yeast protein Npl3p
contains a nonclassical NLS but fails to concentrate in nuclei of
starving cells (7). We therefore propose that the Star sequence
functions as a special nonclassical NLS in starving cells.
-galactosidase in cells
carrying a deletion of NMD5. This might indicate that low
amounts of Star-
-galactosidase enter the nucleus by an
Nmd5p-independent mechanism.
-galactosidase, GFP-Star may accumulate in nuclei by a
special nuclear import pathway that is activated in starving cells (see
model, Fig. 7). However, GFP-Star nuclear
accumulation did not depend on any of the
-importins we have tested.
Specifically, cells lacking a functional NMD5 gene
concentrated GFP-Star in nuclei when entering stationary phase. The
simplest explanation of these results is that nuclear accumulation of
GFP-Star is achieved by nuclear retention. Due to its small size,
GFP-Star diffuses in and out of the nucleus. Once translocated across
the nuclear envelope, the Star sequence may bind to anchors that retain
GFP-Star in nuclei (Fig. 7). On the basis of our results for
Star-
-galactosidase and GFP-Star we propose that the Star sequence
has two functions. First, it supports active nuclear import, a reaction
that depends on Nmd5p. Second, the Star sequence associates with
nuclear anchors that prevent its exit from the nucleus, a reaction
independent of Nmd5p. Both signal functions, i.e. import and
retention, seem to be activated when cells enter stationary phase.
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Fig. 7.
Localization of reporter proteins via the
Star sequence. In early stationary phase cells,
Star- -galactosidase and GFP-Star accumulate in nuclei. Nuclear
accumulation of Star-
-galactosidase requires a specialized nuclear
import pathway that involves Nmd5p. Nuclear retention independent of
Nmd5p contributes to the nuclear accumulation of proteins carrying the
Star sequence, thus preventing nuclear exit of GFP-Star. See
"Discussion" for details.
Nuclear accumulation of GFP-Ssa4p-(16-642) is reversible, indicating that starving cells, when provided with fresh nutrients, export the fusion protein into the cytoplasm. Because GFP-Ssa4p-(16-642) is too large to exit the nucleus by diffusion, nuclear export has to be mediated by an active process. This is in line with our observation that GFP-Ssa4p-(16-642) remains nuclear if cells are transferred to fresh medium but incubated at 4 °C. Moreover, nuclear export of GFP-Ssa4p-(16-642) is sensitive to oxidants that are known to inhibit other nuclear export processes (16).
Our data allow us to propose the following model for protein
localization mediated by the Star sequence (Fig. 7). In logarithmically growing cells a Star-containing protein can be found in the nucleus and
in the cytoplasm. The protein accumulates in nuclei of starving cells,
a process that is mediated by Nmd5p-dependent nuclear
import, with the Star sequence functioning as a special nonclassical
NLS. Furthermore, nuclear retention promoted by the Star sequence
prevents exit into the cytoplasm. At present, we can only speculate
about the role of Ssa4p nuclear concentration in early stationary
phase. Like nuclear accumulation of hsp70s upon heat shock,
concentration of Ssa4p in nuclei may help reduce the denaturation of
proteins in energy-depleted cells. Once provided with fresh nutrients, Ssa4p is released from nuclear anchors and may participate in refolding
of nuclear proteins. Ultimately, Ssa4p nuclear export and shuttling
will localize the protein to both nucleus and cytoplasm as observed in
logarithmically growing cells.
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ACKNOWLEDGEMENTS |
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We thank V. Doye (Paris), D. Goldfarb (Rochester, NY), and G. Schlenstedt (Hamburg, Germany) for providing us with strains and the plasmid encoding NLS-GFP. We are grateful to A. Chu (Montreal, PQ) for critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by grants from Medical Research Council and Natural
Sciences and Engineering Research of Canada, Canada. A scholar of Medical Research Council, Canada and a research scholar
fellow of Fonds de la recherche en santé du Québec.
To whom correspondence should be addressed: Dept. of Physiology, McGill
University, 3655 Drummond St., Montreal, PQ H3G 1Y6, Canada. Tel.:
514-398-2949; Fax: 514-398-7452; E-mail: stochaj@med.mcgill.ca.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M100364200
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ABBREVIATIONS |
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The abbreviations used are:
NLS, nuclear
localization sequence;
-galactosidase, E. coli
-galactosidase;
DAPI, 4',6-diamidino-2-phenylindole;
GFP, A.
victoria green fluorescent protein;
Star, protein sequence
inducing nuclear targeting upon starvation.
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