From the Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, the ¶ Department of Medicine, University Hospitals of Cleveland and The Cleveland Veteran's Affairs Medical Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, and the § Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706
Received for publication, July 21, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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The 70-kDa heat shock proteins are molecular
chaperones that participate in a variety of cellular functions. This
chaperone function is stimulated by interaction with hsp40 proteins.
The Saccharomyces cerevisiae gene encoding the essential
hsp40 homologue, SIS1, appears to function in translation
initiation. Mutations in ribosomal protein L39 (rpl39)
complement loss-of-function mutations in SIS1 as well as
PAB1 (poly(A)-binding protein), suggesting a functional
interaction between these proteins. However, while a direct interaction
between Sis1 and Pab1 is not detectable, both of these proteins
physically interact with the essential Ssa (and not Ssb) family of
hsp70 proteins. This interaction is mediated by the variable C-terminal
domain of Ssa. Subcellular fractionations demonstrate that the binding
of Ssa to ribosomes is dependent upon its C terminus and that its
interaction with Sis1 and Pab1 occurs preferentially on translating
ribosomes. Consistent with a function in translation, depletion of Ssa
protein produces a general translational defect that appears similar to loss of Sis1 and Pab1 function. This translational effect of Ssa appears mediated, at least in part, by its affect on the interaction of
Pab1 with the translation initiation factor, eIF4G, which is dramatically reduced in the absence of functional Ssa protein.
The 70-kDa heat shock proteins are molecular chaperones that
participate in a variety of cellular functions. hsp70 proteins bind
hydrophobic stretches on unfolded proteins, aiding in their folding and
oligomerization and in translocating proteins across membranes
(reviewed in Refs. 1 and 2). This chaperone function of hsp70 is
stimulated by interaction with hsp40 proteins. ATP-bound hsp70 proteins
transiently bind hydrophobic stretches on unfolded substrates, and
their intrinsic ATPase activity is stimulated by hsp40 proteins, which
increases the stability of the hsp70-peptide interaction (1). The
hsp70-hsp40 interaction is conserved from bacteria to humans (2).
In Saccharomyces cerevisiae, there are at least 14 different
hsp70 proteins grouped into five defined subclasses. The two cytosolic
hsp70 subfamilies, SSA and SSB, share 60% amino
acid identity but cannot functionally substitute for one another (2). The essential SSA subfamily contains four members (referred
to collectively as Ssa). Ssa1 and -2 are constitutively expressed, while Ssa3 and -4 are heat-inducible (3). Ssa is important in the
folding and membrane translocation of nascent peptides, nuclear import,
microtubule formation, and the transcriptional response to heat shock
(1, 4-7). The Ssb1 and Ssb2 proteins (referred to collectively as Ssb)
are associated with ribosomes and appear to function in binding nascent
peptides during translation elongation (8, 9). The SSB
subfamily is not essential, but deletion of both members results in a
cold-sensitive phenotype (9). A yeast hsp40 protein, Sis1, is also
ribosome-associated (10). Deletion of SIS1 is lethal, and a
yeast strain that expresses a temperature-sensitive Sis1 protein,
sis1-85, demonstrates a defect in translation
initiation at the nonpermissive temperature (10, 11). Due to its
association with ribosomes, Sis1 has been proposed to function with Ssb
(12). However, Sis1 does not stimulate the ATPase activity of Ssb but
does stimulate that of Ssa (Ref. 13; reviewed in Ref. 14).
The temperature sensitivity of sis1-85 is suppressed by a functional
deletion of ribosome L39 protein (rpl39/spb2) as well as
ribosomal protein L35 (rpl35/sos1), providing further
evidence for a translational function for Sis1 (10). Interestingly, a deletion of rpl39 also suppresses the lethal deletion of
another essential translation factor, poly(A)-binding protein
(PAB1), suggesting that these proteins functionally interact
(15). Pab1 binds the 3'-terminal poly(A) tract within an mRNA and
also plays a role in the stabilization of RNA messages (reviewed in
Ref. 16). Pab1 also serves to bring the 5'- and 3'-ends of mRNA in close proximity by binding the initiation scaffold protein eIF4G (17).
This has been shown to be essential for efficient translation initiation as well as mRNA stability (16, 18, 19).
While the functional significance of the genetic relationship between
PAB1 and SIS1 is unclear, the shared phenotype of
Yeast Methods--
The yeast strains used in this study are
listed in Table I. Yeast cultures
were grown as indicated using either synthetic medium plus 2%
glucose supplemented with the appropriate additives or complete
medium (22). Yeast were grown in the appropriate medium
at either 25 or 30 °C to log phase. Where indicated, yeast were
shifted to 37 °C by resuspending the cell pellet in prewarmed 37 °C medium and incubated at 37 °C for the prescribed time. For depletion experiments, yeast were grown in complete medium with 2%
galactose and then washed three times and shifted to complete medium
with 2% glucose for 7 h before harvesting. The URA3
CEN4 plasmids containing the SSA1 and SSB1
fusion constructs were previously described (12). These were
transformed into JN54 by standard lithium acetate transformation
procedure (22). The C-terminal deletion of SSA was created
by engineering a stop codon by PCR mutagenesis immediately 3' to the
XhoI site used for construction of the chimeras (12).
Fractionation of Ribosomes--
All procedures were performed at
4 °C except where indicated. Yeast cells from 50 ml of log phase
culture were pelleted, treated for 1 min with 10 µg/ml cycloheximide
(Calbiochem), and repelleted. Lysates were made by bead beating the
yeast for 4 min, with intermittent cooling on ice, in polysome buffer
(PB1; 100 mM KCl,
2 mM magnesium acetate, 20 mM HEPES, pH 7.4, 14.4 mM
For cycloheximide treatment of cells, 100 µg/ml cycloheximide was
added to the yeast medium concomitant with a shift to 37 °C.
RNase treatment of the yeast lysate was performed by adding 300 µg/ml
RNase A to the PB, followed by regular lysis. High salt treatment was
performed by centrifuging lysates in a sucrose gradient containing 500 mM KCl. For immunoprecipitation from gradient fractions, lysates were centrifuged through a 10-25% sucrose gradient for 16 h at 23,100 rpm. The polysome pellet was washed and resuspended for immunoprecipitation in sucrose gradient buffer. Nonribosomal fractions (fractions 1 and 2) were collected and directly
immunoprecipitated. Fractions containing the 40, 60, and 80 S ribosomes
were pelleted using an SW50 rotor at 49,700 rpm for 7.5 h. Pellets
were washed and resuspended for immunoprecipitation in sucrose gradient buffer.
Immunoprecipitations--
Lysates were made by resuspending cell
pellets in immunoprecipitation buffer (150 mM KCl, 20 mM Tris, pH 8.0, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1%
Triton, 0.01% SDS) and bead beating for 4 min with intermittent
cooling on ice. Lysates were centrifuged at 15,000 rpm for 10 min at
4 °C. Supernatants were transferred to tubes containing 20 µl
of protein A beads (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) and either 1 µl of HA-12CA5 (D. Templeton, Case Western Reserve University, Cleveland, OH), PAB 1G1 (M. Swanson, University of Florida,
Gainesville, FL), SIS 66932 (23), SSA 1173 (24), or SSB 30733 (9), as
indicated. Where noted, 10 µl of lysate was removed and added to an
equal volume of Laemmli buffer for analysis by Western blot.
Immunoprecipitates were rotated overnight at 4 °C, washed four times
with immunoprecipitation buffer, and resuspended in Laemmli sample
buffer. Immunoprecipitates from gradient fractions were normalized to
10% sucrose by resuspension of the pellets with sucrose gradient
buffer plus 10% sucrose.
Western Blots--
For immunoprecipitates, samples were boiled
for 5 min, and equal amounts of each sample were separated on 10%
SDS-polyacrylamide gel electrophoresis. Proteins were transferred to
Immobilon (Millipore Corp., Bedford, MA) in 0.01 M CAPS and
10% methanol using a semidry electroblotter (Owl Scientific, Woburn,
MA) at 400 mA for 45 min. Immunoprecipitates from the Ssa and Ssb
fusion proteins were separated on an 8% SDS-polyacrylamide gel
electrophoresis containing a 20:1 acrylamide/bisacrylamide ratio. These
gels were transferred to Immobilon for 45 min at 400 mA.
Proteins collected from sucrose gradient fractions were trichloroacetic
acid-precipitated, washed twice in 100 mM Tris, pH 8.0, acetone (1:5, v/v), and solubilized by boiling in Laemmli buffer for 5 min. Pellets from 10-25% gradients were resuspended in polysome
buffer for immunoprecipitation or in Laemmli buffer. Equal amounts of
each fraction were separated on 10% SDS-polyacrylamide gel
electrophoresis and transferred as above to Immobilon (Millipore Corp.).
Membranes were blocked in 5% milk in 1× PBS-T (PBS plus 0.02% Tween
20; Sigma) and probed with the following antibody dilutions in 5%
milk, 1× PBS-T: HA-12CA5 (1:2000), PAB 1G1 (1:5000), SSA 1173 (1:2000,
except in Figs. 1E and 2, where a dilution of 1:600 was
used), SSB 30733 (1:2000, except in Figs. 1E and 2, where a
dilution of 1:600 was used), GST 1:2000 (Amersham Pharmacia Biotech),
SIS 66932 (1:1000), Hsp70 535 (1:1000; E. Craig, University of
Wisconsin, Madison, WI), or eIF4G (1:1000) (17). Western blots were
washed three times for 5 min each in 1× PBS-T. HA-12CA5 and PAB 1G1
antibodies were detected with a goat anti-mouse antibody conjugated to
horseradish peroxidase (ICN Biomedical Inc., Aurora, OH), diluted
1:5000 in 5% milk in 1× PBS-T. SSA 1173, SSB 30733, SIS1 66932, Hsp70
535, and eIF4G antibodies were detected with a goat anti-rabbit
antibody conjugated to horseradish peroxidase (Cappel, Durham, NC)
diluted 1:4000 in 5% milk in 1× PBS-T. GST antibodies were detected
with a rabbit anti-goat antibody conjugated to horseradish peroxidase
(Sigma) diluted 1:8000. Western blots were washed as above and
developed using chemiluminescence with ECL reagents (Amersham Pharmacia Biotech).
Slot Blots--
RNA was extracted according to standard
procedures from 17 A600 of ssa1-45
yeast or wild-type yeast grown at 25 °C and 30 min following shift
to 37 °C (22). Volumes throughout the RNA extraction were kept
equivalent so that the final RNA content represented RNA extracted from
an equivalent A600 of yeast. Equal volumes of
RNA were diluted as indicated in diethylpyrocarbonated-treated water, 50% formamide, 6.5% formaldehyde, 5X SSC, and bound to nitrocellulose (Schleicher and Schuell) using the Minifold II slot blot
apparatus (Schleicher and Schuell). Total mRNA abundance was
determined by probing with cDNA made from whole cell RNA from SSA cells as previously described (22, 25). Blots were
developed by autoradiography.
35S in Vivo Labeling--
Four 10-ml cultures of
SSA and ssa1-45 yeast were grown at 25 °C
overnight until mid-log phase. At zero time, half of the cultures were
centrifuged and resuspended in YEPD prewarmed to 37 °C. At 0, 10, 30, and 60 min, individual cultures were labeled with 10 µCi of
[35S]methionine (1175 Ci/mmol; PerkinElmer Life Sciences)
for 2 min. Triplicate samples of 0.5 ml were removed, an equal volume
of ice-cold 25% trichloroacetic acid was added, and the samples were vortexed. Samples were stored on ice for 30 min, and the precipitated proteins were collected onto Whatman GF/C glass fiber filters by vacuum
filtration and washed with 5% trichloroacetic acid and 95% ethanol.
Amino acid incorporation into acid-precipitable material was determined
by scintillation counting in a Beckman LS6000 SE liquid scintillation
counter. Final values were corrected for A600 readings.
In Vitro Binding Studies--
Bacterial strains containing
histidine-tagged Pab1 (26), pGEX2TSSA1-RI, or pGEX2T-A1C1 Pab1 and Sis1 Interact with the hsp70 Protein
Ssa--
PAB1 and SIS1 share a genetic
interaction with RPL39, and their gene products are
ribosome-associated proteins that function in translation. Therefore,
we investigated whether Pab1 and Sis1 proteins interacted, as
determined by coimmunoprecipitation. Immunoprecipitations were
performed from the wild type yeast strain JN54 (herein called SSA) using either a Pab1 antibody or Sis1 antibody (Fig.
1). The immunoprecipitates were analyzed
by Western blot with either the Sis1 (Fig. 1A) or Pab1 (Fig.
1B) antibodies. Pab1 was not detected in the Sis1
immunoprecipitate, and similarly, Sis1 was not detected in the Pab1
immunoprecipitates. Thus, at the limit of resolution of these
immunoprecipitations, Sis1 and Pab1 do not directly interact.
hsp40 proteins function together with an hsp70 partner, and interaction
between Sis1 and Pab1 may be indirect and mediated via an hsp70
partner. Therefore, coimmunoprecipitation studies were performed to
determine whether Sis1 or Pab1 bound either Ssb or Ssa.
Immunoprecipitations were performed and analyzed as above using either
Sis1 (Fig. 1A), Pab1 (Fig. 1B), Ssa-specific (Fig. 1C), or Ssb-specific antibodies (Fig. 1D).
These experiments demonstrated that Ssa, but not Ssb,
coimmunoprecipitated with Sis1, and similarly Pab1 coimmunoprecipitated
Ssa but little detectable Ssb. In reciprocal immunoprecipitations, Pab1
coimmunoprecipitated with Ssa but not Ssb (data not shown). To ensure
that the differential association of Ssa and Ssb with Pab1 was not due
to differences in total abundance of these hsp70 homologues, an aliquot
of lysate was analyzed by Western blotting for Ssa and Ssb, concurrent
with the Pab1 immunoprecipitates (Fig. 1E). As demonstrated
here, the Pab1-Ssa interaction appears to be 8-10-fold stronger than
the Pab1-Ssb interaction (as determined by densitometry) when the total
amount of these proteins is taken into account. This preferential binding of Ssa to Pab1 was confirmed by further studies (Fig. 2 and data not shown). Thus, there
appears to be a specific interaction of Ssa hsp70 proteins with Pab1
and Sis1.
To determine whether Pab1 bound all Ssa family members,
immunoprecipitations were performed with lysates from strains lacking different family members: SSA; JN49, which lacks Ssa1 and
Ssa2 (herein called ssa1ssa2 (29)); and JB67, whose only
source of Ssa is a temperature-sensitive SSA1 (herein called
ssa1-45) (Fig. 1F). Pab1 coimmunoprecipitated
with Ssa from both the ssa1ssa2 and ssa1-45
strains, indicating that Pab1 binds to Ssa family members 1, 3, and 4. Although we do not have definitive data on Ssa2, it is likely that Pab1
also binds this protein.
Pab1 Binds to the C-terminal Domain of Ssa--
To identify the
Pab1 binding site on Ssa, we took advantage of the fact that Pab1 did
not bind Ssb and analyzed Ssa/Ssb fusion proteins by
immunoprecipitation. Each fusion contains an ATPase domain, a peptide
binding domain, and a C-terminal domain from one of these two hsp70
protein subclasses (12). Each fusion is named for the domains they
contain (i.e. AAB contains the ATPase and peptide binding
domain from Ssa1 and the variable domain from Ssb1). Pab1 was
immunoprecipitated from SSA yeast transformed with one of
the six fusion constructs, and immunoprecipitates were analyzed by
Western blot using either Ssa (Fig. 2A) or Ssb (Fig.
2B) antibodies. As a protein expression control, aliquots of
each of the lysates were analyzed by Western blotting (Figs. 2,
A, lanes 1-3, and B,
lanes 1-4). All six of the fusion proteins, as
well as the native hsp70, were expressed to similar levels and were
recognized by the appropriate antibody. The variable C-terminal domain
of Ssa was necessary and sufficient for Pab1 association, as seen by
the coimmunoprecipitation of BBA, and not AAB, with Pab1 (Fig.
2A, lane 6). In contrast, fusion
proteins containing the C terminus of Ssb only weakly
coimmunoprecipitated with Pab1, despite the fact that all of these
proteins were expressed at similar levels (Fig. 2B,
lanes 5-8). We assume that the ABA fusion
protein also coimmunoprecipitated with Pab1, but because it comigrated
with wild type Ssa, the association of ABA with Pab1 could not be
assessed (12).
The above experiments demonstrated a specific interaction between the
C-terminal domain of Ssa and Pab1 in vivo. To confirm that
this was due to a direct interaction between these two proteins and not
simply coprecipitation with a common ribonucleoprotein complex, an Ssa
C-terminal fragment was fused to GST, expressed in bacteria, purified
on a glutathione column, and eluted. As a control, GST was used.
Recombinant histidine-tagged Pab1 was purified, and eluates from the
GST or GST-Ssa purification were applied to either metal affinity resin
alone or metal affinity resin with immobilized Pab1 (Fig.
2C). Additionally, these pull-downs were either treated with
RNase A or left untreated. GST alone did not bind to immobilized Pab1,
but the C-terminal fragment of Ssa bound Pab1 in the presence or
absence of RNase A treatment. In addition, another GST-Ssa fragment,
encompassing a shorter C-terminal fragment, also bound to immobilized
Pab1 to a similar degree (data not shown). As demonstrated here, the
Pab1-Ssa interaction is not dependent on RNA but appears due to a
direct protein-protein interaction between Pab1 and Ssa. Additionally,
neither the addition of excess poly(A) mRNA to
immunoprecipitates nor in vivo depletion of poly(A)
mRNA blocks the Ssa-Pab1 association (data not shown). Finally,
since Ssa and Pab1 coimmunoprecipitate from cytosolic fractions that do
not contain detectable mRNA or ribosomes (see Fig. 4B
and data not shown), the interaction of these two proteins appears direct.
Ssa Proteins Are Found in Association with Translating
Ribosomes--
Since both Sis1 and Pab1 are ribosome-associated
proteins and interact with Ssa, we determined whether Ssa associated
with ribosomes. Lysates from SSA yeast were analyzed by
sedimentation through 10-50% sucrose gradients, and the distribution
of ribosomes in the gradient was determined by absorbance at 254 nm.
Proteins were recovered from the fractionated lysate, and the presence of Ssa in the fractions was analyzed by Western blotting using antibodies that recognized Ssa proteins but not Ssb. As seen in Fig.
3A, the Ssa proteins
(top panel) were most abundant in the top of the
gradient (fractions 1 and 2). However, a significant portion sedimented
at the same position as ribosomal subunits and translating polysomes.
This is comparable with that seen with Ssb, Sis1, and Pab1 (Fig.
3A, lower panels).
Previous experiments suggested that the most rapidly sedimenting Ssa
proteins were not bound to ribosomes, since their sedimentation was
unaffected by RNase A (12). To determine whether the Ssa proteins found
in the polysome fractions were polysome-associated, lysates from
SSA yeast were treated with RNase A prior to fractionation. This reduced the amount of material that sedimented in polysome fractions and increased the amount in 80 S fractions (Fig. 3, compare
A and B). Coincident with this, the amount of Ssa
protein was reduced in polysomal fractions, paralleling results for
Ssb, Sis1, and Pab1 (Fig. 3B, lower
panels). Thus, under the conditions used here, the rapidly
sedimenting Ssa proteins are part of an RNase-sensitive particle.
As an independent means of confirming Ssa association with polysomes,
strains temperature-sensitive for translation initiation factors
prt1-1 (F294) and eIF4E (cdc33) were utilized
(Fig. 3C and data not shown). Under permissive conditions,
Pab1, Sis1, Ssa, and Ssb exhibit a polysomal distribution, similar to
that seen in the SSA yeast (data not shown). At the
nonpermissive temperature (37 °C for 30 min), both of these strains
showed a loss of polysomes and accumulation of 80 S ribosomes. A
concomitant loss of Ssa from the polysome fractions was also observed,
similar to the results for Pab1, Sis1, and Ssb (Fig. 3C).
Association of Ssa, as well as the other proteins, with polysomes was
stable to sedimentation in 500 mM KCl (Fig. 3D),
suggesting that all of these proteins are present on translating ribosomes.
The C-terminal domain of Ssa appears required for its interaction with
Pab1. To determine whether this domain also mediated Ssa association
with polysomes, a C-terminal truncation was created at the juncture
between the peptide binding domain and the C terminus as defined in the
chimeras (12). This truncated protein was expressed in SSA
yeast and total extract, nonpolysomal, and polysomal fractions
isolated. Ssa Interacts with Sis1 and Pab1 on Translating Polysomes but Not
on 40 S Subunits or 80 S Ribosome Couples--
While both Sis1 and Ssa
are abundant in the cytoplasm and associate with ribosomes, if Ssa
functions as the hsp70 partner for the translational functions of Sis1
then their interaction should be detectable on ribosomes. To determine
whether this was the case, cell lysates from SIS1-HA yeast
were fractionated through 10-25% sucrose gradients. Polysomes and
associated proteins are found in pellets, while the cytoplasm and 40, 60, and 80 S fractions remain in the supernatant. While interaction of
Sis1 with Ssa was observed in the most slowly sedimenting subribosomal
fraction, the interaction appeared strongest in polysomes, as judged by the amount of coimmunoprecipitating material (Fig.
4A). In addition, since both
Ssa and Sis1 are present in 80 S fractions, the absence of a detectable
interaction in this fraction provides evidence that the Sis1-Ssa
interaction is specific for translating ribosomes and not simply due to
coprecipitation with ribosomes (30).
Similar studies were performed to characterize the site of Pab1
interaction with Ssa. Using the SSA strain, Pab1
immunoprecipitates from subribosomal, polysomal, 40 S, 60 S, and 80 S
fractions were analyzed by Western blotting with the Ssa and Pab1
antibodies (Fig. 4B). While Ssa and Pab1 interacted in the
subribosomal fraction, there was an equivalent interaction on
polysomes. Similar to the Ssa-Sis1 interaction, the Ssa-Pab1
interaction was not detected on either ribosomal subunits or in
translationally inactive 80 S ribosomes despite the fact that these
proteins were present in these fractions (see also Fig. 3A)
(30). Thus, physical interaction of Ssa with both Pab1 and Sis1 appears
to occur preferentially on translating ribosomes.
Depletion of Ssa Affects Translation--
The
temperature-sensitive mutant, sis1-85, exhibits a
translational defect (10). Because Sis1 activates the ATPase activity of Ssa and we have shown that Ssa interacts with Sis1 on translating ribosomes, we hypothesized that Ssa was the hsp70 partner to Sis1 in
translation initiation. To determine whether Ssa had a role in
translation, we utilized the temperature-sensitive ssa1-45 yeast strain. Incorporation of [35S]methionine into newly
translated proteins was measured during a 2-min pulse label at 0, 10, 30, and 60 min after shift to the nonpermissive temperature (Fig.
5A). While in the wild type
SSA strain a minor decrease in translation rate occurred at
10 and 30 min (reduced to 84 and 71%, respectively, of wild type),
complete recovery occurred by 60 min postshift. In contrast,
translation rate was significantly reduced by 10 min postshift in the
ssa1-45 strain (reduced to 62% as compared with the
control ssa1-45 at 25 °C) and continued to decrease to
9.7% of control at 60 min postshift.
Since heat shock affects transcription and has been implicated to play
a role in mRNA stability as well (31-33), we determined whether
the decrease in translation rate was due to a decrease in mRNA
abundance. RNA was extracted from equivalent amounts of ssa1-45 yeast at the permissive temperature and 30 min
following temperature shift. Total cellular mRNA was quantified by
slot-blot hybridization with 32P-labeled cDNA made from
total cellular mRNA. As demonstrated in Fig. 5B, there
was no significant decrease in mRNA abundance following temperature
shift. Therefore, the reduction in translation seen at this time
appears due to an effect on the translational machinery and not a lack
of mRNA.
Sucrose gradients were used to further define the nature of the
translational defect in the ssa1-45 yeast. (Fig.
5C). At the permissive temperature, the SSA and
ssa1-45 strains had a similar distribution of ribosomes.
However, following shift to the nonpermissive temperature, the
ssa1-45 strain demonstrated a rapid loss of polysomes with
an accompanying increase in 80 S ribosomes. The 60% decrease in
polysome content at this time paralleled the decrease in translation rate, as determined by radioactive methionine incorporation (Fig. 5A). With additional time of incubation, a further decrease
occurred, and by 60 min the loss of polysomes was nearly complete (Fig. 5C and data not shown). These results are similar to that
seen with Sis1 depletion, suggesting that these proteins both affect translation initiation (10).
To ensure that the sustained decrease in translation observed following
temperature shift of the ssa1-45 strain was not simply an
exaggerated heat shock response, Ssa protein was depleted by use of a
strain that contained a triple SSA1,2,4 knockout covered by
a plasmid that expressed SSA1 under the control of the
galactose promoter (herein called pGAL-SSA1) (34). Following
7 h of growth in glucose medium, translation was assessed as
above. Similar to the ssa1-45 strain, a reduction in
polysome content occurred, and this was accompanied by an increase in
80 S ribosomes (Fig. 5D). This change is made more dramatic
by the fact that shifting yeast from galactose to glucose increases
translation rate, with an accompanying increase in polysomes and a
decrease in 80 S ribosomes (Ref. 35 and data not shown).
The effect of Ssa depletion on the relative abundance of polysomes and
80 S ribosomes was consistent with a defect in translation initiation
(10, 36). If Ssa depletion preferentially affected initiation, then
cycloheximide should prevent the redistribution of ribosomes into
inactive 80 S complexes by increasing the elongation phase of
translation or by blocking run-off of translating polysomes. As
demonstrated in Fig. 5E, this was the case, since concurrent exposure to cycloheximide at the nonpermissive temperature prevented polysome depletion and 80 S ribosome accumulation. Thus, the primary translational defect of Ssa depletion appears to be in initiation.
Depletion of Ssa Affects Pab1 Association with eIF4G--
The
translational effects of Pab1 are mediated at least in part by its
interaction with initiation factor, eIF4G (16-19). We therefore
investigated whether Ssa affected this interaction by analyzing Pab1
immunoprecipitates for eIF4G by Western blotting (Fig.
6). Growth at the nonpermissive
temperature did not affect interaction of eIF4G with Pab1 in the
wild-type SSA strain (Fig. 6, top two
panels). However, in the ssa1-45 strain, growth
at the nonpermissive temperature resulted in a substantial decrease in
recovery of eIF4G. Although expression of eIF4G was also reduced in
this latter strain, growth at the nonpermissive temperature did not
alter eIF4G expression. Thus, interaction of Pab1 and eIF4G is reduced
in the absence of functional Ssa.
The data presented here demonstrates that the yeast hsp70
homologue, Ssa, has a specific physical interaction on translating polysomes with Pab1, as well as with Sis1. As shown previously for
mammalian hsp40 and hsp70 and shown here for Pab1-Ssa, these interactions are mediated by the variable C-terminal domain on hsp70
(20). Ssa appears necessary for translation initiation, since a
temperature-sensitive strain of Ssa shows a decrease in polysome
content and an accumulation of salt-labile, 80 S ribosomes at the
nonpermissive temperature. Depletion of Ssa also reduces interaction of
Pab1 and eIF4G, suggesting this to be a target for the chaperone
activity of Ssa.
The data presented here provide evidence that Ssa interacts with Pab1.
One explanation for this is that Ssa serves as a chaperone for Pab1.
However, in that case, Ssa would be expected to interact with Pab1 via
its peptide binding domain. Instead, Pab1 binds to the C-terminal
variable domain on Ssa. Further, since Sis1 does not bind Pab1, Pab1
appears to be an unlikely substrate for the chaperone activity of the
Ssa-Sis1 complex. Hop, another protein that binds to the C-terminal
variable domain on hsp70s, stimulates the interaction between
substrate-bound hsp70 and hsp90 (20, 37-39). Therefore, Pab1 may
function like Hop to mediate the association of Ssa and its partner
Sis1 with substrates on ribosomal complexes.
Since Sis1 stimulates the ATPase activity of Ssa, it was not surprising
that these proteins coimmunoprecipitated (10, 13). In fact, as
demonstrated here, this interaction appears markedly enhanced on
polysomes, suggesting that Ssa is the hsp70 partner for previously
described translational function of Sis1. However, despite the fact
that genetic evidence suggests a functional relationship between Sis1
and Pab1 (10, 15), a physical interaction of these two proteins could
not be demonstrated. A Pab1-Sis1 complex may be transient or
undetectable by our means. Alternatively, Sis1 and Pab1 may bind Ssa
competitively through the Ssa C-terminal domain (20). In addition to
its role in stimulating the ATPase activity of hsp70 proteins, the
hsp40 partner functions in the substrate specificity and intracellular
localization of hsp70 (40-42). Therefore, by binding both Pab1 and
Sis1 through its C-terminal domain, Ssa may be first targeted to
ribosomal complexes through its interaction with Pab1 and subsequently
to a specific substrate by Sis1.
The fact that Sis1 and Pab1 both affect translation initiation and that
Ssa associates with Pab1 and Sis1 on ribosomes, led us to investigate
whether this latter protein also affected translational initiation.
Such a role for Ssa was foreshadowed in experiments investigating the
role of Ssa in folding newly synthesized ornithine transcarbamylase
(7). Incubation of the ssa1-45 strain at the nonpermissive
temperature resulted in a 90% decrease in ornithine transcarbamylase
translation. Utilizing this same temperature-sensitive strain, the
experiments reported here demonstrate a similar overall decrease in
protein synthesis rate, a reduction in polysomes, and an increase in
translationally inactive 80 S ribosomes at the nonpermissive
temperature. These results are consistent with a defect in translation
initiation. The translational effect of depletion of Ssa is similar to
those previously reported following loss of function of both Sis1 and
Pab1 (10, 15). Thus, these interacting proteins all function in
translation initiation.
Both Ssb and Ssa associate in an RNase-sensitive, salt-stable manner
with ribosomes, and both aid in the folding of nascent peptides (1, 8,
9). However, while Ssa and Ssb are related subfamilies of cytosolic
hsp70 proteins, they do not complement each other, and SSA
function is essential, while SSB is not (9, 12, 29). Sis1
stimulates the ATPase activity of Ssa but Ssb (13). This work provides
further evidence that Ssa and Ssb have distinct functions. These
proteins differ dramatically in their ability to associate with Pab1.
Depletion of Ssa and Ssb produce dissimilar effects upon translation as
assayed by polysome profiles (Refs. 9 and 12 and this study). Current
data suggest a role for Ssb in elongation, whereas our data suggest a
role in initiation for Ssa (Refs. 9 and 12 and this study). Consistent
with this, subtle differences in the polysomal distribution of these two proteins exist, with Ssa being most abundant in small polysomes, while Ssb is most abundant in the largest polysomes. Interestingly, the
bacterial translation initiation factor IF2 has chaperone function
similar to hsp70 (43), suggesting that chaperone functions may be
required at multiple steps during translation.
Pab1 is part of a multiprotein complex that plays a role in translation
initiation, and one of its functions is to bind eIF4G, bringing the 5'-
and 3'-ends of the mRNA in proximity (16-19). While mutation of
the Pab1 binding site on eIF4G is not lethal, this interaction is
presumed to be important for cap-dependent translation
initiation (19, 26, 44). The data presented here demonstrate that Ssa
appears necessary for the association of Pab1 with eIF4G. Although we
have not yet been able to unequivocally demonstrate a physical
interaction between eIF4G and Ssa (data not shown), this result
provides one explanation for the effect of Ssa depletion on translation
initiation. However, it may be anticipated that Ssa modulates other, as
yet unidentified ribosomal functions (possibly related to rpl39), since
the translational effect of heat shock is conserved in bacteria, in
which the mechanism of mRNA binding to ribosome is different and
does not involve eIF4G.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pab1 and sis1-85 genes suggests a potential
interaction between the two in translation initiation. However, as
demonstrated here, there is no detectable physical association between
Pab1 and Sis1. In contrast, Ssa interacts with both Sis1 and Pab1, and
these interactions are specific for the Ssa subfamily of hsp70s. As previously shown for the interaction between hsp40 and hsp70, the
interaction of Ssa with Pab1 is mediated via the variable C-terminal
domain of Ssa rather than the peptide binding domain (20). Like Sis1,
Ssa associates with ribosomes, and a temperature-sensitive strain of
SSA (ssa1-45) demonstrates a severe
translational defect (10, 21). Depletion of Ssa decreases the
association of Pab1 with eIF4G, suggesting that the translational
effect of Ssa is mediated at least in part by its effect on the
interaction of these two proteins.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Yeast strains
-mercaptoethanol, 100 µg/ml cycloheximide).
This mixture was centrifuged at 5000 rpm for 8 min, and the supernatant
was removed. 5-10 A254 units were loaded
onto a 16.2-ml 10-50% sucrose gradient containing 100 mM
KCl, 5 mM MgCl2, 20 mM HEPES, pH
7.4, and 2 mM dithiothreitol. Lysates were sedimented in a
Beckman SW28.1 rotor at 27,000 rpm for 4.5 h. Gradients were
collected with continuous monitoring at 254 nm using an ISCO UA-5
absorbance detector and 1640 gradient collector. Samples run over
10-25% gradients were sedimented in a Beckman SW28.1 rotor at 20,000 rpm for 16.5 h. Pellets were resuspended in polysome buffer, and
supernatant fractions were either collected as above or pooled where indicated.
B1 (E. Craig, University of Wisconsin, Madison, WI) were grown overnight at
37 °C. The His-Pab1 was purified as previously described (27). The
GST fusion proteins were purified as previously described (28) and
eluted from glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech)
in 10 mM glutathione (Sigma). The eluate was incubated with
either Talon metal affinity resin (CLONTECH, Palo
Alto, CA) alone or His-Pab1 immobilized on Talon resin for 1 h at
4 °C. The resin was washed three times in wash buffer (100 mM KCl, 50 mM Tris, pH 8.0, 1 mM
phenylmethylsulfonyl fluoride, 0.1% SDS, 0.1% Triton X-100, 100 µM bovine serum albumin, 10% glycerol, 2 mM
imidazole). Samples were then either treated or mock-treated with 2 µg of RNase A for 20 min at 26 °C. Samples were washed twice, and
the resin was resuspended in wash buffer with 200 mM
imidazole. His-Pab1 was eluted for 15 min at 26 °C, and the eluate
was removed. The eluate was separated on 12% SDS-polyacrylamide gel
electrophoresis and analyzed by Western blotting.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Ssa interacts with Sis1 and Pab1.
A, Ssa1 coimmunoprecipitates Sis. Lysates were prepared from
SSA yeast. Proteins were immunoprecipitated (IP)
with protein A beads or beads plus Pab1, Ssa, or Ssb antibodies.
Immunoprecipitates were probed for Sis1 by immunoblotting.
B, Ssa coimmunoprecipitates Pab1. Proteins were
immunoprecipitated using Ssa, Sis1, or Ssb antibodies and analyzed for
Pab1 as described above. C, Pab1 and Sis1
coimmunoprecipitate Ssa. Proteins were immunoprecipitated using Pab1 or
Sis1 antibodies and probed for the presence of Ssa as described above.
D, Ssb does not coimmunoprecipitate with Pab1 or Sis1.
Proteins were immunoprecipitated with Ssb, Pab1, or Sis1 antibodies and
probed for Ssb as described above. E, Pab1
immunoprecipitates Ssa and not Ssb. Proteins were immunoprecipitated
with Pab1 antibody and probed for Ssa or Ssb as described above. An
aliquot of the lysate was used as a control for protein expression and
loaded in lanes 1 and 4. F,
multiple members of the Ssa family coimmunoprecipitate Pab1. Lysates
from the SSA, ssa1ssa2, and ssa1-45
yeast were immunoprecipitated with the Ssa antibody and probed for the
presence of Pab1 as described above.
View larger version (27K):
[in a new window]
Fig. 2.
The interaction between Pab1 and hsp70
proteins is specific to the Ssa C terminus. A, fusion
proteins containing the C terminus of Ssa coimmunoprecipitate with
Pab1. Lysates from the SSA strain transformed with either
the ABA, BAA, or BBA fusion protein
constructs were immunoprecipitated with the Pab1 antibody or beads
alone as a control. Prior to immunoprecipitation, an aliquot of the
lysate was removed as a control for protein expression (left
panel). Immunoprecipitates (middle
panel) and control immunoprecipitates (right
panel) were probed for Ssa by immunoblotting. All
figures represent equal exposure times. B, fusion
proteins containing the C terminus of Ssb do not coimmunoprecipitate
with Pab1. Lysates from the SSA strain transformed with
either the BAB, ABB, or AAB fusion
protein constructs were immunoprecipitated with Pab1 antibody. Prior to
immunoprecipitation, an aliquot of the lysate was removed as a control
for protein expression (left panel).
Immunoprecipitates were probed for Ssb by immunoblotting
(right panel). The right
panel represents a 10-fold longer exposure time than the
left panel. C, His-Pab1 associates
with a recombinant C-terminal fragment of Ssa. Recombinant Ssa fusion
proteins were purified and eluted from glutathione beads and incubated
with immobilized histidine-tagged Pab1 or resin alone. Samples were
either mock- or RNase A-treated and then eluted with 200 mM
imidazole from the resin and analyzed by Western blotting
(WB) with anti-GST antibody.
View larger version (23K):
[in a new window]
Fig. 3.
Ssa proteins are associated with
ribosomes. A, Ssa associates with polysomes. Lysate
prepared from SSA yeast was fractionated through a sucrose
gradient and collected with continuous monitoring of UV absorbance at
254 nm. The positions of the 40, 60, and 80 S peaks are indicated.
P indicates the presence of the first polysome fraction.
Total proteins from each fraction were collected and probed for the
presence of either Ssa, Ssb, Pab1, or Sis1 by immunoblotting. Blots are
displayed in the lower panels. B, Ssa
association with polysomes is RNase-sensitive. Lysate was treated with
RNase A prior to sedimentation. Fractionated proteins were collected as
above and probed for the presence of Ssa, Ssb, Pab1, and Sis1 by
immunoblotting. C, Ssa abundance in polysome fractions is
directly related to polysome content. The prt1-1 strain was
grown at 25 °C until mid-log phase and then shifted to 37 °C for
30 min. Proteins from gradient fractions were collected and probed for
Ssa, Ssb, Pab1, and Sis1 as described above. D, Ssa
association with polysomes is stable to 500 mM KCl. Lysate
from SSA yeast was fractionated through sucrose gradients
containing 500 mM KCl. The presence of Ssa, Ssb, and Pab1
in the gradient fractions was determined by immunoblotting.
E, the C terminus of Ssa is required for its interaction
with polysomes. Lysates prepared from SSA yeast or
SSA yeast transformed with a C-terminal deletion of
SSA were loaded onto 10-25% sucrose gradients.
Nonpolysomal fractions were combined, and 1% of the total extract, 5%
of the nonpolysome fraction, and 20% of the pelleted polysomes were
probed for Ssa using the hsp70 antibody described under "Experimental
Procedures."
C terminus Ssa was identified in these fractions by
Western blotting (Fig. 3E). In contrast to the full-length Ssa, the C-terminal truncation of Ssa dramatically reduced its association with polysomes. Thus, an intact C terminus is required not
only for efficient binding of Ssa to Pab1 but also for the association
of Ssa with polysomes.
View larger version (33K):
[in a new window]
Fig. 4.
Ssa associates with Sis1 and Pab1 in both
nonribosomal and polysomal fractions. A, Ssa
coimmunoprecipitates with Sis1 from polysomes. Lysates prepared from
the SIS1-HA strain were fractionated in 10-25% sucrose
gradients. Proteins were collected from the soluble cytoplasm
(CYTO), 40 S, 60 S, 80 S, and polysome (POLYS)
fractions; immunoprecipitated (IP) with the HA antibody
(top and third panels), Ssa antibody
(second panel), or beads alone (bottom
panel); and probed for the presence of Sis1 (top
panel) or Ssa (panels 2-4). The
bottom two panels represent equal
exposure times. B, Ssa coimmunoprecipitates with Pab1 from
polysomes. Lysates prepared from the SSA strain were
fractionated onto 10-25% sucrose gradients. Proteins were collected
from the soluble cytoplasm (CYTO), 40 S, 60 S, 80 S, and
polysome (POLYS) fractions. Proteins were mmunoprecipitated
with Pab1 antibody (top and middle
panels) or beads alone (bottom panel)
and probed for Pab1 (top panel) or Ssa
(middle and bottom panels) as
described above. The middle and bottom
panels represent equal exposure times. WB,
Western blot.
View larger version (32K):
[in a new window]
Fig. 5.
Depletion of Ssa causes a defect in
translation. A, [35S]methionine
incorporation into acid-precipitable peptides. SSA (squares)
and ssa1-45 (circles) yeast were pulse-labeled
for 2 min with [35S]methionine at 25 °C and at 10, 30, or 60 min following shift to 37 °C. Values are in
cpm/min/A600. B, mRNA content in
ssa1-45 yeast. ssa1-45 yeast were split into two equal
aliquots and grown at 25 °C or 37 °C for 30 min. RNA was
extracted from an equal A600 of each sample, and
recovered RNA was loaded on a slot blot at the indicated dilutions. The
blot was probed with 32P-cDNA made from total mRNA.
C, ssa1-45 yeast demonstrate a decrease in polysomes and an
increase in 80 S ribosomes at the nonpermissive temperature.
SSA and ssa1-45 yeast were grown at 25 °C in
YEPD. Half of the culture was shifted to 37 °C for 30 min. Equal
amounts of lysate were prepared and loaded onto sucrose gradients.
Gradients were collected with continuous monitoring of UV absorbance at
254 nm. The positions of the 40, 60, and 80 S peaks are indicated.
P indicates the presence of the first polysome fraction.
D, Ssa depletion inhibits translation. pGAL-SSA
yeast were grown in medium containing 2% galactose until log phase.
Half of the culture was shifted to medium containing 2% glucose for
7 h. Equal amounts of lysate were analyzed by sucrose gradient
sedimentation as described above. E, cycloheximide prevents
the accumulation of 80 S ribosomes and reduction of polysomes.
ssa1-45 yeast were treated with cycloheximide at 25 or
37 °C for 30 min. Lysates were analyzed by sucrose gradient
sedimentation, as above.
View larger version (59K):
[in a new window]
Fig. 6.
Depletion of Ssa affects the eIF4G
interaction with Pab1. SSA and ssa1-45
yeast were grown at 25 °C, and then half of the culture was shifted
to 37 °C for 30 min. Equal amounts of lysate were prepared and
immunoprecipitated with Pab1 antibody (top
panels) or beads alone (bottom panel).
Recovered proteins were analyzed by immunoblotting with antibodies to
either eIF4G (top panel) or Pab1
(second panel). Expression of eIF4G in the cell
lines was assessed by removing an aliquot of the lysate prior to
immunoprecipitation and probing for the presence of eIF4G as described
under "Experimental Procedures" (third
panel).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Carl Stratton, Christine Pfund, and members of the Bill Merrick laboratory for technical assistance and helpful discussion and Dr. Alan Sachs, Dr. Allan Jacobsen, Dr. Bill Merrick, and Carl Stratton for critical reading of the manuscript. We also thank Drs. Alan Sachs, Maurice Swanson, Dennis Templeton, Kim Arndt, Alan Hinnebusch, and Nahum Sonenberg for providing useful yeast strains, clones, and antibodies.
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FOOTNOTES |
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* This work was supported in part by a grant from the American Cancer Society, Cuyahoga Unit (to J. O. H.) and by National Institutes of Health Grant RO1 GM31107 (to E. A. C.).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 National Institutes of Health Grants HK07147-21A1 and DK09915.
To whom correspondence should be addressed: Dept. of
Hematology and Oncology, 10900 Euclid Ave., BRB309B, Cleveland, OH
44106-4937. Tel.: 216-368-8758; Fax: 216-368-1166; E-mail:
joh2@po.cwru.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M100266200
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ABBREVIATIONS |
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The abbreviations used are: PB, polysome buffer; CAPS, 3-(cyclohexylamino)propanesulfonic acid; GST, glutathione S-transferase; HA, hemagglutinin.
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