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INTRODUCTION |
The import of proteins into nuclei is mediated by soluble nuclear
localization signal (NLS)1
receptors. SV40 large T-antigen-like NLSs are bound in the cytoplasm by
karyopherin
(Kap
), which serves as an adapter to link NLS-cargo to karyopherin
(Kap
). Kap
mediates docking at the
cytoplasmic face of the nuclear pore complex (1). Saccharomyces
cerevisiae contains a single Kap
gene encoded by
SRP1. Translocation of the NLS-cargo-Kap
/
ternary
complex occurs through the central channel of the nuclear pore complex.
Once in the nucleus, the NLS-cargo dissociates and Kap
and
are
recycled to the cytoplasm (1, 2). Only a portion of the cellular import
traffic is mediated by Kap
/
heterodimers. Other classes of
NLS-cargo, for example shuttling pre-mRNA binding proteins that
display M9-type import signals (3), are transported by Kap
-like
factors that bind directly to cognate NLS-cargo without the aid of Kap
adapters (1, 4).
Hsp70/Hsc70s (collectively referred to here as Hsp70s) are conserved
molecular chaperones that participate in a variety of cellular
functions, including protein folding and transport and the repair of
stress-induced damage (5-7). Hsp70s are composed of a 44-kDa
N-terminal ATPase domain, an 18-kDa peptide binding domain, and a
C-terminal 10-kDa variable domain of unknown function (8, 9). S. cerevisiae contains two families of cytosolic Hsp70 genes,
SSA1-4 and SSB1-2 (10, 11).
The yeast Ssa-type Hsp70s are similar to the cytosolic Hsp70s found in
other organisms including bacteria. To date, Ssb-type Hsp70s have been
identified only in fungi. In S. cerevisiae, Ssb1 and Ssb2
are associated with translating ribosomes and can be cross-linked to
nascent polypeptides (12, 13).
Hsp70s have been proposed to function in NLS-directed nuclear transport
by promoting the formation and stability of NLS-cargo-Kap
complexes
(reviewed in Refs. 1, 14, and 15). Thus, the ectopic expression of
human Hsp70 in mouse cells rescued the import of a protein carrying a
mutant NLS (16). Conversely, the elevated expression of SSA1
in yeast suppressed a transport defect in srp1-31 cells
(15). Microinjected antibodies against Hsc70 inhibited NLS-directed
import (17), and the depletion of Hsp70 from cytosolic extracts
inhibited import in cell-free assays (18-20). Finally, the finding
that the nuclear localization of Hsp70 is itself dependent on the
co-import of NLS-cargo (19) suggests that Hsp70 is imported in
association with the NLS-cargo-Kap
/
ternary complex.
In the present study we delineate a functional nuclear export signal
(NES) in the C-terminal domain of Ssb1p and show that it is responsible
both for the different subcellular localizations of Ssa1p and Ssb1p and
for their differential function in NLS-directed nuclear transport.
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MATERIALS AND METHODS |
Strains, Plasmids, and Culture Conditions--
Except where
noted, all yeast strains used in this study were based in a W303
(strain WHY12) genetic background (MATa
ade2-1 leu2-3, 112 his3-11,
and 15 trp1-1 ura3-1
can1-100). Construction of strains containing
nup188-
(21) and xpo1-1 alleles
(22) and L40 cells (23) were previously described. DNA manipulations were performed using standard protocols (24). The construction of
pGAD-NLSGFP and pGFP-URA3 (25), YCpGAL1-SSA1, and YcpGAL1-SSB1, which
contain the SSA1 and SSB1 genes under the control
of the GAL1 inducible promoter (6), and the
SSA1/SSB1 chimeric plasmids (26) were previously
described. Chimeras that divided the ATPase domain into two halves
according to crystal structure (8) were constructed using an
EagI site introduced by polymerase chain reaction at the
gene sequence corresponding to amino acid 177 in Ssa1p and amino acid
181 in Ssb1p. A BamHI site introduced by polymerase chain
reaction at the N terminus of Ssa1 and Ssb1 was used to clone the
chimeras into pCUG2, placing them under GAL1 control. pCUG2
was constructed by inserting a 690-base pair EcoRI/BamHI fragment containing the
GAL1/GAL10 promoter into the pRS316 (27). pGFP-N-FUS plasmid
(28), containing the MET25 promoter and GFP, was used to
express GFP-Sssa1p/Ssb1p fusions for localization studies. In
pGFP-pA-Ca/Cb plasmids, protein A synthetic analog, the so-called
Z-domain (29), was inserted between the GFP and C-terminal
sequences of SSA1 and SSB1. Appropriate cloning
sites were created by high fidelity polymerase chain reaction. Polymerase chain reaction-amplified fragments and vector junctions in
completed plasmid constructs were verified by DNA sequencing. Standard
complete or selective synthetic media were used as described (30).
NLS-GFP Import Assay, Galactose Induction, and Heat
Shock--
NLS-GFP kinetic import assays were performed as described
(15) with minor modifications as indicated in the text. For heat shock,
cells grown at 30 °C were pelleted, resuspended in 39 °C pre-warmed medium, and incubated at 39 °C for 1 h. Control
cells were resuspended in 30 °C pre-warmed medium and incubated at
30 °C for 1 h. Induction of protein expression from pGAL1
vectors was achieved by resuspending glucose-grown cells in synthetic medium containing 2% galactose and incubating in a shaking water bath
at 30 °C for 2 h.
Fluorescent Conventional and Confocal Microscopy--
Only fresh
exponentially growing cultures were used for fluorescence microscopy
study. Fluorescence microscopy was performed using an Olympus BH-2
microscope, and pictures were taken with a mounted Olympus C-35AD-2
camera. Kodak TMAX 400 film was processed with TMAX developer,
Indicator stop bath, and Rapid fixer (Eastman Kodak Co.). Confocal
images were obtained with a Leica TCS NT microscope, and digital images
were processed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA).
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RESULTS |
Nucleocytoplasmic Localization of GFP-Ssa1p and GFP-Ssb1p--
The
subcellular localization of Ssa1p and Ssb1p was determined using GFP
fusions. GFP-Ssa1p and GFP-Ssb1p fusion proteins were expressed from a
MET25 inducible promoter in WHY12 cells and
localized by confocal microscopy. As expected, both Hsp70s were
localized in the cytoplasm, but only GFP-Ssa1p accumulated to any
significant degree in the nuclei (Fig.
1). Both fusions were excluded from
vacuoles. The localization of GFP-Ssa1p in both the cytoplasm and
nucleus is consistent with the distribution of Ssa-type Hsp70s in other
eukaryotes (31, 32). In contrast, the exclusive cytoplasmic
localization of GFP-Ssb1p is unique among known cytosolic Hsp70s.

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Fig. 1.
Subcellular localization of GFP-Ssa1p and
GFP-Ssb1p. Localization of GFP-Ssa1p (A, E) and
GFP-Ssb1p (C, G) by GFP fluorescence and nuclei (B, D,
F, H) by Hoechst staining. Cells were maintained at 30 °C
(A-D) or heat-shocked at 42 °C for 1 h
(E-H). Arrows point to the
nuclei.
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A feature of the stress response in many eukaryotes is the migration of
cytoplasmic Ssa-type Hsp70s from the cytoplasm into the nucleus where
they accumulate to high levels (32, 33). To determine whether this
phenomenon occurs in S. cerevisiae, the localization of
GFP-Ssa1p and GFP-Ssb1p was observed following heat shock treatment.
After a 60-min heat shock treatment, nuclear GFP-Ssa1p levels
increased, relative to cytoplasmic levels, but not to a great extent
(Fig. 1). Heat shock had no detectable affect on the localization of
GFP-Ssb1p (Fig. 1).
The C Terminus of Ssb1p Directs Cytoplasmic
Localization--
Hsp70s, including Ssa1p and Ssb1p, can be divided
into three structural domains: a 44-kDa ATPase domain, an 18-kDa
peptide binding domain, and a 10-kDa C-terminal variable domain (Fig. 2A). The ATPase domain can be
further divided into two subdomains (8). We constructed chimeric genes
containing a mixture of Ssa and Ssb structural domains, most of which
are designated by a three-letter code (Ref. 26 and this study). For
example, "ABA" contains the Ssa ATPase domain, the Ssb peptide
binding domain, and the Ssa C-terminal domain. Chimeras that contain
subdivided ATPase domains are designated, for example, "(AB)AA" or
"(BA)AA." Chimeras that lack particular domains are designated
"-AA," which, in this example, lacks an ATPase domain. For the
purpose of localization studies, each chimera was expressed as a GFP
fusion protein. GFP was not fused to the Hsp70 constructs for the
purpose of functional studies (see below).

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Fig. 2.
Subcellular localization of Ssa1p/Ssb1p
chimeras. A, Ssa1p and Ssb1p are divided into three
functional domains, the ATPase, peptide binding, and C terminus. Thus,
Ssa1p is designated AAA. In some constructs the ATPase
domain was divided in half such that Ssa1p would be indicated by
(AA)AA. B, GFP-Ssa1p/Ssb1p chimeras,
their masses, and their cellular localizations:
N C, exclusion from nuclei; N = C, approximate equilibration; N > C, obvious nuclear accumulation. C, fluorescent
images of selected GFP-Ssa1p/Ssb1p fusions and nuclei in the same group
of cells positioned by Hoechst staining (column H).
Arrows indicate the positions of nuclei in matched
cells.
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Localization of the various GFP-Ssa1p/Ssb1p chimeras was determined by
conventional and confocal fluorescence microscopy, and the fluorescence
in cells was scored as being either equally distributed between nucleus
and cytoplasm (N = C), exclusively cytoplasmic (N
C), or
significantly more concentrated in the nuclei (N > C) (Fig. 2,
B and C). These data indicate that the Ssb1p
C-terminal domain is responsible for the low nuclear levels of
GFP-Ssb1p. Thus, ABB, (AB)BB, (BA)BB, AAB, BAB, and -BB all showed
background levels of nuclear staining. In contrast, (AB)AA, BAA,
(BA)AA, ABA, and BBA localized to both the cytoplasm and the nucleus
(Fig. 2B). The localizations of selected chimeras are shown
in Fig. 2C. Immunoblot analysis of cell extracts expressing these GFP fusion proteins indicates that they are stable (data not
shown). In addition to these data, GFP fused to AA- and BB- localized
to both the cytoplasm and the nucleus (Fig. 2B). It is worth
noting here that a previous study (34) implicated the C terminus of rat
Hsc70 in its bidirectional trafficking.
To determine whether the Ssb1p C terminus can function autonomously to
prevent nuclear accumulation, Ssa1p ("Ca") and Ssb1p ("Cb")
C-terminal domains were fused to the C terminus of a GFP-protein A
(GFP-pA) reporter protein (Fig.
3A) and localized in cells by confocal microscopy (Fig. 3B). Whereas GFP-pA-Ca (53 kDa)
localized both to the nucleus and the cytoplasm, GFP-pA-Cb (51 kDa) did not stain the nuclei (Fig. 3B). Immunoblot analysis
demonstrated that these fusions were stable in vivo (not
shown). We conclude that the C terminus of Ssb1p can function
autonomously to prevent nuclear localization. The Ssb1p C terminus
could function either as a cytoplasmic retention sequence to exclude
Ssb1p from the nucleus or as a NES to direct the export of any Ssb1p
that reaches the nucleus.

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Fig. 3.
Role of Ssb1p C-terminal domain in
subcellular localization. A, diagram of GFP-protein
A-Ssa1p/Ssb1p C terminus reporter proteins
(GFP-pA-Ca and
GFP-pA-Cb). B, subcellular
localization of GFP-pA-Ca and GFP-pA-Cb (GFP) and
corresponding nuclei (H). C, delineation of the
Ssb1p cytoplasmic localization sequence. GFP-Ssb1p reporter proteins
(full-length Ssb1p contains 613 amino acids (aa)) containing
the indicated C-terminal truncations and internal deletion were
localized by fluorescence microscopy and scored as being completely
excluded from nuclei (C N), somewhat
excluded (C > N), or approximately
equilibrated (C = N). Alignment of putative
SSB-type proteins from Saccharomyces cerevisiae
(S.C), Candida albicans (C.A),
Schizosaccharomyces pombe (S.P), and
Kluyeromyces marxianus (K.M.) shows
identical (asterisks) and similar (dots)
residues. Residues that correspond to the consensus NES motif are
indicated in bold.
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The minimal Ssb1p C-terminal sequence necessary to prevent nuclear
localization was delineated by truncation and deletion analysis of a
GFP-Ssb1p reporter protein (Fig. 3C). These results point to
a short sequence that contains a match to the consensus sequence for
leucine-rich NESs (35, 36). Ssb1p homologs from other fungi also
contain this NES motif (Fig. 3C).
Ssbp Contains a Functional NES--
An in vivo assay
for NES function based on the approach of Fritz and Green (37) was
developed in the Wente laboratory (Washington University, St. Louis,
MO) and was generously provided to us prior to publication. In this
assay, HIS3 is transcribed only when a plasmid-borne
transcriptional activator, composed of the LexA DNA binding domain and
the pseudorabies activation domain, is expressed in the nucleus. Cells
that express this activator grow on medium lacking histidine (Fig.
4). When HIV-1 Rev or Gle1p NESs were
inserted between the LexA and pseudorabies domains, the cells failed to
grow on selective medium (Fig. 4), presumably because the activator was
exported before it could activate HIS3 transcription. Cells
expressing an activator with a mutant Rev NES grew well on selective
medium (Fig. 4). Significantly, cells expressing activators containing
the entire C-terminal domain of Ssb1p (amino acids 546-613), or just
the putative NES (amino acids 574-587), failed to grow on selective
medium (Fig. 4). These data support the hypothesis that Ssb1p residues
574-587 function as a NES.

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Fig. 4.
Functional inhibition of a transcriptional
transactivator by the Ssb1p NES. Growth of cells on medium lacking
histidine is assessed for cells expressing transactivator alone
(no NES) or transactivator fused to wild-type or mutant Rev
NESs, Gle1p NES, Ssb1p C terminus (546-613), or
Ssb1p NES consensus sequence (574-587).
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The export of leucine-rich NES-cargo is mediated by a Kap
-like
protein called exportin, encoded in yeast by
XPO1/CRM1 (21). xpo1-1
cells exhibit reduced NES-directed export at nonpermissive temperatures. For example, a protein kinase inhibitor NES-GFP-NLS fusion protein, which is normally efficiently exported from nuclei, becomes almost exclusively nuclear in xpo1-1
cells shifted to 37 °C (22). Fig. 5
shows the localization of GFP-Ssb1p in XPO1 and
xpo1-1 cells following a shift from 30° to
40 °C. GFP-Ssb1p was exclusively cytoplasmic in XPO1
cells after 60 min at 40 °C. In contrast, GFP-Ssb1p fluorescence
appeared in the nuclei of xpo1-1 cells within 20 min at 40 °C and became equilibrated across the nuclear envelope
within 60 min (Fig. 5). As expected, the GFP-Ssb1p was exclusively
cytoplasmic in xpo1-1 cells carrying XPO1 on a plasmid (Fig. 5). We conclude that Ssb1p is
exported from nuclei in a Xpo1p-dependent fashion. The
accumulation of GFP-Ssb1p in the nuclei of xpo1-1
cells at a nonpermissive temperature (Fig. 5, panels F and
I) indicates that Ssb1p normally shuttles between the
nucleus and the cytoplasm.

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Fig. 5.
Localization of GFP-Ssb1p in
temperature-sensitive xpo1-1
cells. GFP-Ssb1p was localized by fluorescence microscopy in
wild-type and xpo1- cells expressing XP01 or
xpo1-1 at 30 °C (A, B, C), after 20 min at
40 °C (D, E, F), and after 60 min at 40 °C (G,
H, I). Arrows point to the representative nuclei.
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NLS-GFP Nuclear Localization Defect of nup188-
Cells Is
Suppressed by Heat Shock and SSA1 Induction--
NLS-GFP is a small
nuclear reporter protein that can be used to monitor nuclear transport
kinetics in wild-type and mutant yeast cells using method proposed
before (15). nup188-
cells, which are
morphologically normal and double at wild-type rates (not shown),
accumulate abnormally high cytoplasmic levels of NLS-GFP (Fig.
6A, panel B). We
previously showed that the NLS-GFP import defect of
srp1-31 cells was suppressed either by heat shock or the induction of GAL1-SSA1. As shown in Fig.
6A, panel D, the induction of
GAL1-SSA1 expression also suppressed the steady
state NLS-GFP localization defect of nup188-
cells. In
contrast, induction of GAL1-SSB1 did not suppress
the steady state nup188-
defect (Fig. 6A,
panel F). The level of Hsp70 gene expression under these conditions was determined by immunoblot using specific polyclonal antibodies (26) and revealed that the concentration of Hsp70s increased
3-4-fold following GAL1 induction (data not shown). The
SSA1 induction upon heat shock was comparable with
GAL1-SSA1 induction (data not shown).

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Fig. 6.
Induction of SSA1 but not
SSB1 suppresses the NLS-GFP nuclear localization
defect of nup188- cells.
A, NLS-GFP expressing normal (NUP188) and mutant
(nup188- ) cells were observed before and after induction
of SSA1 and SSB1 expression (see "Materials and
Methods"). B, SSA1 induction stimulates NLS-GFP
import kinetics in nup188- cells. 37 °C import time
courses are shown for NLS-GFP import in nup188- cells
that were untreated (closed circles), heat-shocked
(HS) for 1 h (open triangles), or induced
for GAL1-SSA1 (closed squares) or
GAL1-SSB1 expression for 2 h (open
circles) and wild type, (wt) cells (open
squares). wild- type (wt). C, extended time
course of NLS-GFP import at 37 °C in NUP188
(circles) and nup188- (squares)
cells at 37 °C.
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The effects of heat shock and GAL1-SSA1 expression on
NLS-GFP re-import kinetics in nup188-
cells grown at
30 °C and assayed at 37 °C are shown in Fig. 6B.
NLS-GFP import in NUP188 cells proceeded rapidly and to
virtual completion within 5 min. In contrast, NLS-GFP import in
nup188-
cells proceeded with biphasic kinetics, beginning
with a "burst" phase, during which 20-40% of the cells rapidly
accumulated NLS-GFP, followed by a slower quasi-plateau phase (see Fig.
6C). We do not know whether the biphasic nature of these kinetics are a
property of all nup188-
mutated cells or, alternatively,
indicate the presence of different cell populations, perhaps differing
in their Hsp70 content. nup188-
cells that were
heat-shocked, or GAL1-SSA1-expressing cells,
showed wild-type import kinetics (Fig. 6B). The induction of
GAL1-SSB1 expression in nup188-
cells resulted
in a slight, but reproducible, increase in the fraction of cells
showing good import during the burst phase (Fig. 6B).
nup188-
cells grown at 37 °C showed better nuclear
accumulation of NLS-GFP than cells grown at 30 °C (not shown).
37 °C is a heat shock temperature, and cells grown at this
temperature contain high levels of Ssa-type Hsp70s (17). For this
reason we extended the 37 °C NLS-GFP import time course to allow for the full development of a heat shock response. An extended 37 °C
time course revealed that after the initial burst phase NLS-GFP nuclear
accumulation continued slowly until virtually all of the cells in the
culture showed good nuclear localization (Fig. 6C). The
duration of the slow phase roughly corresponds to the rate at which
Hsp70 accumulates in cells during heat shock. When assayed at 30 °C,
which is not a heat shock temperature, the kinetics of NLS-GFP import
began with a burst but quickly reached a plateau of between 20-40%
nuclear cells that was stable for at least 3 h (not shown). These
results suggest that the slow increase in NLS-GFP nuclear localization
at 37 °C is the result of the heat shock induction of Ssa-type Hsp70
gene expression. We conclude that elevated levels of Ssa1p are
sufficient to suppress both the steady state and kinetic NLS-GFP
localization defects of nup188-
cells.
Nuclear Localization of Ssa1p/Ssb1p Chimeras Correlates with Their
Ability to Suppress the nup188-
Nuclear Transport Defect--
The
efficient NES-directed export of Ssb1p could provide a basis for the
inability of Ssb1p to suppress the NLS-GFP nuclear localization defect
in nup188-
cells. To test this hypothesis we
assayed NLS-GFP import kinetics in nup188-
cells
expressing various Ssa1p/Ssb1p chimeras and plotted the "% nuclear
cells" after a 15-min re-import (Fig.
7). The Ssa1p/Ssb1p chimeras for this
functional analysis were not fused to GFP. As shown in Fig. 7, the
expression of chimeras containing the C terminus of Ssb1p (BBB, ABB,
and AAB) failed to suppress the NLS-GFP localization defect. In
contrast, chimeras containing the C terminus of Ssa1p (AAA, BAA, and
BBA), and truncations of Ssa1p and Ssb1p lacking either C-terminal
domain (AA- and BB-), suppressed the nup188-
NLS-GFP
import defect. We conclude that the C-terminal domain of Ssb1p, which
contains a functional NES, is a dominant inhibitor of Hsp70 function in
NLS-directed nuclear transport. Together with the GFP-Ssa1p/Ssb1p
localization data shown in Fig. 2B, these experiments
demonstrate a strong positive correlation between the steady state
nuclear localization of Hsp70 and its functions in NLS-directed nuclear
transport.

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Fig. 7.
Effect of Ssa1p/Ssb1p chimeras on NLS-GFP
import in nup188- cells.
Various SSA/SSB chimeras, indicated by three-letter codes (see
"Results"), were cloned into pCUG2 and expressed in NLS-GFP
expressing nup188- cells. NLS-GFP import assays were
performed at 37 °C as described under "Materials and Methods."
The percentages of nuclear cells were scored at T = 15 min.
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DISCUSSION |
The aim of this study was to investigate the role of Hsp70 in
NLS-directed nuclear transport and, more specifically, to elucidate the
molecular basis for the differential function of Ssa- and Ssb-type
Hsp70s. We conclude that an NES in the divergent 8 kDa C-terminal
domain of Ssb1p is necessary and sufficient for the low nuclear levels
of Ssb1p and for its inability to stimulate NLS-directed nuclear
transport. Therefore, the differential function of Ssa1p and Ssb1p in
nuclear transport is due to the NES-directed export of the Ssb1p and
not to functional differences in their ATPase or peptide binding
domains. Previous work showed that certain Ssa1p/Ssb1p chimeras,
containing either the ATPase or peptide binding domains of Ssa1p, could
rescue phenotypes associated with the double deletion of
SSB1 and SSB2 (26). Thus, Ssa1p domains can
function in Ssb1p-mediated processes. Here, we show that the peptide
binding and ATPase domains of Ssb1p can function in a Ssa1p-mediated
process. This is not to say that Ssa1p can replace Ssb1p function
in vivo or vice versa. There are no known examples in which
one class of Hsp70 can completely replace the function of a second
class. Although a truncated Ssb1p that lacks its NES still functions in
nuclear transport, it could not rescue the depletion of all four
SSA gene products. In this regard, yeast mitochondrial
Hsp70, loaded into reconstituted endoplasmic reticulum vesicles, could
not replace endoplasmic reticulum Hsp70 in an in vitro
protein secretion assay (39). The inability of mitochondrial Hsp70 to
function in BiP-mediated secretion was attributed to the inability of
mitochondrial Hsp70 to associate productively with Sec63p, a
BiP-specific DnaJ co-chaperone (39). The possible role of Hsp70
co-chaperones in nuclear transport has not been explored.
The very existence of the Ssb1p NES, and the accumulation of GFP-Ssb1p
in the nuclei of xpo1-1 cells, suggests that
Ssb1p normally shuttles between the nucleus and cytoplasm. Although Ssa-type Hsp70s have previously been shown to shuttle between the
nucleus and the cytoplasm in Xenopus oocytes (40), Ssb-type Hsp70s have been assumed to function exclusively in the cytoplasm (12,
13). These new results suggest that Ssb1p may function in the nucleus
as well as in the cytoplasm.
Elevated Levels of Ssa1p but Not Ssb1p Stimulate NLS-directed
Nuclear Transport--
A 2-3-fold increase in cellular Ssa1p levels
suppressed the kinetic and steady state NLS-GFP nuclear localization
defects of nup188-
cells. The severity of the
nup188-
defect, and its complete suppression, provided a
clean, specific in vivo assay for Hsp70 function in nuclear
transport. The NLS-GFP nuclear transport defects of strains containing
temperature-conditional mutations in other transport factors,
specifically srp1-31 and
nup82-3 cells (15), were also suppressed by
GAL1-SSA1 induction, but these defects are less
severe and are not, therefore, ideally suited to quantification. The
present analysis is restricted to the role of Hsp70 in the import of
SV40-type NLSs and does not address the potential involvement, or lack
thereof, of Hsp70 in the transport of other classes of import or export
cargo (4).
The stimulatory effects of GAL1-SSA1 induction on
NLS-GFP import suggest that Hsp70 functions by stimulating at least one rate-limiting step along the nuclear import pathway. This result implies that the concentration of Ssa-type Hsp70s under non-stress conditions is normally limiting for nuclear transport and that GAL1-SSA1 or stress induction generates an
incipient pool of Hsp70 that is available to interact with the nuclear
transport apparatus. Under stress conditions in higher eukaryotes,
cytosolic pools of Hsp70 are recruited into the nucleus (31, 33).
Therefore, in addition to an increase of Hsp70 levels as a result of
new synthesis, there is an additive increase in nuclear levels because of compartment redistribution. In yeast, we observed a minor but reproducible increase in relative nuclear GFP-Ssa1p fluorescence following heat shock. Although this result is consistent with a minor
redistribution of Hsp70 in yeast, it is possible that under heat shock
conditions nuclear levels remained constant and only appeared to
increase because cytoplasmic levels decreased through turnover.
It is not known whether Hsp70 is absolutely required for the import of
NLS-cargo or, alternatively, whether it functions as an enhancer of
nuclear transport efficiency. Although the literature is undecided on
this issue, a strict requirement for Hsp70 in the nuclear transport of
selected NLS-cargo was demonstrated using isolated nuclei and
permeabilized cell assays, under which circumstances endogenous Hsp70
levels can be more or less completely depleted (17-20).
GFP-Ssa1p Is Localized Both in the Nucleus and the Cytoplasm,
Whereas GFP-Ssb1p Is Cytoplasmic--
The localization of GFP-Ssa1p to
both the nucleus and the cytoplasm is consistent with a large body of
evidence on the localization of Ssa-type Hsp70s in a variety of
organisms and cells. The exclusive cytoplasmic localization of
GFP-Ssb1p is unique for non-organelle Hsp70s. A technical weakness in
this analysis of Hsp70 localization is its reliance on the use of GFP
fusions, which could produce localization artifacts. The validity of
the GFP fusion localization results reported here, however, have been
corroborated by two lines of evidence. First, the ability of various
Ssa1p/Ssb1p chimeras and truncations to suppress the
nup188-
NLS-GFP nuclear localization defect is correlated
perfectly with the localization of the GFP-Ssa1/Ssb1 chimeras. Those
chimeras that localized to nuclei suppressed the transport defect,
whereas the chimeras that failed to suppress the transport defect also
failed to accumulate in nuclei. It is essential to note that the
functional assays were performed with Ssa1p/Ssb1p chimeras that were
not fused to GFP. Second, the key finding of the GFP-based localization
studies was the existence of the Ssb1p leucin-rich NES, which in other
experiments was shown to function as an NES out of the context of a GFP
fusion protein.
Both genetic and biochemical evidence suggest that Ssb1p and Ssb2p are
functionally interchangeable and have primary roles in protein
synthesis (13, 41). A large fraction of Ssbp co-sediments with
translating ribosomes, and
ssb1
ssb2 cells
are cold- and hygromycin B-sensitive (12). Why then does Ssb1p contain
an NES? The presence of an NES in a cytoplasmic protein suggests that
the protein shuttles between the nucleus and the cytoplasm and, more
interestingly, has a nuclear function. The Ssb1p NES is not required
for any known Ssb1p function. An Ssa1p/Ssb1p chimera containing the
ATPase and peptide binding domains of Ssb1p and the Ssa1p C-terminal
domain, and hence lacking the NES, rescued both
ssb1
ssb2 phenotypes (26). This experiment did not, however, prevent access of Ssb1p to the nucleus.
Ssb1p could serve any number of functions in the nucleus, a few of
which are mentioned here. Because Ssb1p associates with ribosomes (12)
and Ssa-type Hsp70s have been observed to localize in the nucleus
preferentially to the nucleolus, it is possible that Ssb1p functions in
some aspect of ribosome biogenesis. SSB1 was identified in a
high copy number screen for suppressors of a mutation in a proteasome
subunit (42, 43). Thus, Ssb1p may participate in proteasome-mediated
protein degradation. The proteasome complex is localized both in the
cytoplasm and the nucleus (44), so it is possible that Ssb1p interacts
transiently with a nuclear proteasome pool. In mammalian cells, a link
between the heat shock response and proteasome activity has been noted.
Specifically, the expression of a transcriptional regulator of the heat
shock response, HSF2, is activated in response to proteasome
inhibitors (38). Hsp70 has also been implicated in the autoregulation
of the heat shock response in mammalian cells. Both Hsp70 and the co-chaperone Hdj1p bind Hsf1p and together repress heat shock gene
transcription (45). By analogy, Ssb1p could function in the nucleus as
a regulator of gene expression.
With the discovery of the Ssb1p NES, it is apparent now that both Ssa1p
and Ssb1p shuttle across the nuclear envelope. Like Kap
and
,
Hsp70 would be expected to shuttle during its role in NLS-directed
nuclear transport. Thus, it is unclear why the Ssb1p NES would inhibit
its function in transport. The induction of
GAL1-SSB1 expression did stimulate a small but
reproducible increase in NLS-GFP import in nup188-
cells
(Fig. 6B). It is possible that Ssb1p might promote the
targeting and translocation of NLS-cargo during its shuttling cycle,
but upon entering the nucleus, most of the Ssb1p-associated NLS-cargo
complex might be exported before the NLS-cargo could be released to the
nucleoplasm. Also, it is possible that a significant steady state
concentration of nuclear Hsp70 is required for its function in nuclear
transport. For example, Hsp70 may be involved in the recycling of
transport factors such as Kap
and Kap
back to the cytoplasm.
Normal nuclear levels of Ssb1p may be too low, or simply unavailable, to facilitate factor recycling.
Role of the Hsp70 Chaperone System in NLS-directed Nuclear
Transport--
Molecular genetic evidence supports the notion that
Hsp70 facilitates the formation and stability of the NLS-Kap
complex (15, 16). Cell biological evidence indicates that Hsp70 is co-imported with the NLS-cargo-Kap
/
ternary complex (19, 46).
Furthermore, Hsp70s bind with high affinity to bona fide NLS
peptides (47, 48) and NLS-like peptides (49, 50). Standard models of
Hsp70 chaperone action predict that cycles of NLS peptide binding and
release could function to minimize nonspecific intermolecular
interactions between the NLS and cellular constituents. The chaperone
cycle could, in effect, increase the concentration of free NLS-cargo
available for Kap
binding. Hsp70s normally work in conjunction with
co-chaperones that facilitate and regulate peptide binding/release and
ATPase activity, and it will be interesting to investigate the
potential role of co-chaperones in nuclear transport.