(Received for publication, October 17, 1995; and in revised form, February 7, 1996)
From the
The 46-kDa protein YDJ1 is one of several known yeast homologues
of the Escherichia coli DnaJ protein. Like all J homologues,
it shares homology with the highly conserved NH-terminal
``J-domain'' of DnaJ. A component of the DnaK (Hsp70)
chaperone machinery that mediates protein folding, DnaJ is necessary
for survival at elevated temperatures. It stimulates ATP hydrolysis by
DnaK and effects the release of DnaK-bound polypeptides. Previous
genetic and biochemical studies indicate that the J-domain is necessary
for these functions. Using peptides corresponding to J-domain sequence,
we show that a peptide containing the highly conserved His-Pro-Asp
sequence at positions 34-36 in the J-domain competes off YDJ1
stimulation of Hsp70 ATPase activity. Inhibitory concentrations of
peptide do not prevent binding of folding substrates, therefore YDJ1
must interact with Hsp70 at a site distinct from that for substrate
binding. This interaction is critical for Hsp70 activity, since a
mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates
neither Hsp70 ATPase nor substrate release. The importance of the
proper function of this region of the protein is supported by the poor
growth and temperature-sensitive phenotype of yeast expressing ydj1Q34.
The heat shock family of proteins (Hsps) ()include
members now known to function as molecular chaperones. These Hsps bind
nascent polypeptides as they emerge from cytosolic ribosomes (Beckmann et al., 1990; Nelson et al., 1992), escort fully
translated proteins to their destinations in cellular organelles,
maintain them in a transport-competent partially unfolded state
(Deshaies, 1988), and are necessary on both sides of the membrane for
the efficient transport of proteins into mitochondria (Cheng et
al., 1989; Kang et al., 1990; Caplan et al.,
1992a) and endoplasmic reticulum (Vogel et al., 1990; Scherer et al., 1990; Nguyen et al., 1991). The roles of
chaperones in protein folding have been extensively studied (for recent
review, see Hartl et al.(1992)). There are two major chaperone
families identified as mediators in protein folding. First, the Hsp70s
bind short (7-9-amino acid) sequences of extended polypeptides
(Flynn et al., 1991). Second, the Hsp60s bind to emergent
secondary structures on folding proteins (Landry and Gierasch, 1991).
Neither the Hsp70s nor the Hsp60s act alone; they require
co-chaperones, which modulate their activities (Georgopoulos et
al., 1990).
YDJ1 is a co-chaperone protein in Saccharomyces
cerevisiae which modulates the activity of Hsp70 (Cyr et
al., 1992). It shares extensive homology to the Escherichia
coli protein DnaJ and is one of several homologues of DnaJ
identified in yeast. Each of the homologues has evolved to occupy a
specific niche in the eukaryotic cell. Whereas YDJ1 is largely
cytosolic and endoplasmic reticulum membrane-associated (Caplan and
Douglas, 1991), SIS1 is predominantly nuclear and cytosolic
ribosome-associated (Luke et al., 1991), SCJ1 is found in the
endoplasmic reticulum matrix (Schlenstedt et al., 1995), MDJ1
is associated with the mitochondrial inner membrane (Rowley et
al., 1994), SEC63 is an endoplasmic reticulum integral membrane
protein (Sadler et al., 1989), Zuotin is nuclear and possesses
DNA binding properties (Zhang et al., 1992), and CAJ1 is
membrane-associated and appears to bind calmodulin (Mukai et
al., 1994). Another J homologue, XDJ1, is either a silent gene or
transcribed under unknown conditions (Schwarz et al., 1994).
The common feature of all of these proteins which defines them as J
homologues is homology to the NH-terminal 80 amino acids of
DnaJ protein. This sequence is defined as the J-domain. Whereas SEC63,
Zuotin, and CAJ1 share only this J-domain, the other yeast homologues,
including YDJ1, share homology and structural features with DnaJ
elsewhere in the protein as well (Caplan et al., 1993).
Presumably, each eukaryotic J homologue is specialized to perform,
within its cellular environment, one or a few of the many known
functions of DnaJ. Of the yeast homologues, YDJ1 and SCJ1 are most
closely related to DnaJ (Caplan et al., 1993). However, YDJ1
is distinguished among the homologues by being farnesylated (Caplan et al., 1992b).
The prototype J homologue, DnaJ, was first
identified as a gene product necessary for -phage replication in E. coli and has been cloned and sequenced (Bardwell et
al., 1986; Ohki et al., 1986). Since its initial
characterization, DnaJ has been shown to act in conjunction with the E. coli Hsp70 prototype, DnaK, and a third protein, GrpE.
Together, this trio participates in such diverse functions as
-phage replication (Osipiuk et al., 1993; Hoffmann et
al., 1992), plasmid P1 replication (Sozhamannan and Chattoraj,
1993; Wickner et al., 1992), chromosomal DNA replication (Hupp
and Kaguni, 1993), folding of nascent polypeptides (Hendrick et
al., 1993), export of fully translated polypeptides from the
bacterium (Wild et al., 1992), the repair of heat-induced
protein damage (Schroder et al., 1993; Ziemienowicz et
al., 1993), and the assembly of macromolecular complexes in
flagellum synthesis (Shi et al., 1992). In the folding of
nascent polypeptides and denatured proteins, DnaK binds and releases
extended hydrophobic regions, preventing protein misfolding (Langer et al., 1992). Each cycle of binding and release is dependent
upon ATP hydrolysis, at which DnaK is slow. DnaJ stimulates the ATP
hydrolytic activity of DnaK, allowing a completed cycle of peptide
binding and release, whereas GrpE acts as a nucleotide exchanger,
promoting continued cycles of activity (Liberek et al.,
1991a). Interestingly, DnaJ also possesses chaperone ability in its own
right; a recent report suggests that DnaJ binds polypeptides first,
recruiting DnaK for subsequent binding (Henrick et al., 1993).
Recent genetic and biochemical evidence supports the long standing
idea that the conserved J-domain of DnaJ and its homologues mediates
interaction with DnaK and cognate Hsp70s. Mutations in this region
prevent function of SEC63 in conjunction with the endoplasmic reticulum
Hsp70, Kar2 (Scidmore et al., 1993). In E. coli,
characterization of the dnaJ259 mutant that cannot support
-phage replication (Sell et al., 1990) revealed a single
amino acid change in a highly conserved region within the J-domain. The
NH
-terminal 108 amino acids of DnaJ alone, containing the
full J-domain, are sufficient to support
-phage replication and to
stimulate DnaK ATP hydrolysis (Wall et al., 1994).
Previous work from this laboratory demonstrated that YDJ1 stimulates the ATPase activity of and polypeptide substrate release from its most likely cytosolic cognate Hsp70, SSA1 (Cyr et al., 1992). In the present study, we have used synthetic peptides corresponding to J-domain sequence to compete with purified YDJ1 protein to ask which regions specifically interact with Hsp70 to stimulate these activities. One such region was identified, mutagenized, and tested in for in vivo effects and in vitro activity.
Nine
peptides corresponding to the YDJ1 sequence (Caplan and Douglas, 1991)
were synthesized. Four 20-mers, p1-20, p21-40,
p41-60, and p61-80, span the NH-terminal 80
amino acids of YDJ1. Additionally, pYHPD, X
YHPDX
, and
YHPDX
correspond to amino acids 33-36,
30-39, and 33-52, respectively. p21-40H34Q sequence
was identical to p21-40 with the exception that glutamine was
substituted for histidine at position 34. Two peptides containing the
YDJ1 COOH-terminal sequence were designated pSASQ and pC-farnesyl.
pSASQ was a 13-mer comprised of the COOH-terminal amino acids encoded
by the YDJ1 nucleotide sequence, with the exception that serine was
substituted for cysteine at amino acid 406. pC-farnesyl was a 10-mer
corresponding to the COOH terminus of the mature protein and was
farnesylated before use in competition experiments.
Farnesylation of
pC-farnesyl was performed chemically. ()A 1.2 molar ratio of
farnesyl bromide (Aldrich) diluted in 2 µl of dimethyl sulfoxide
was added to 0.3 mg of peptide dissolved in 70% CH
CN, 70
mM NaHCO
. The reaction proceeded for 4 h at 4
°C, after which farnesylated peptide was separated from
nonfarnesylated peptide, farnesyl bromide, and farnesyl hydroxide by
reverse phase HPLC (10-mm
25-cm Selectosil C
, 300
Å, Phenomenex) using a gradient of 32-100%
CH
CN, 0.1% trifluoroacetic acid, over 70 min. pC-farnesyl
eluted at 43 min. The column eluent was monitored at 254 nm.
YDJ1 and ydj1Q34 were
purified as described previously (Cyr et al., 1992) from
BL21(DE3) E. coli (Novagen, Madison, WI) containing pET9dYDJ1
(Caplan et al., 1992b) and pET9dYQPD, respectively. Cells were
grown in LB + kanamycin at 37 °C until A = 1.0. Isopropyl-1-thio-
-D-galactopyranoside
was added to 0.5 mM, and induction proceeded for 2 h, after
which cells were harvested and resuspended in ice-cold 20 mM MOPS, pH 7.5, 10 mM DTT, 0.5 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride, 10.0 µM leupeptin, and
10.0 µM pepstatin. After cell disruption by sonication,
the lysate was cleared by centrifugation at 100,000
g and loaded onto DE52 equilibrated with lysis buffer. The column
was washed with 3 column volumes of buffer, and elution was performed
using a 0-500 mM NaCl gradient over 20 volumes. Eluted
protein was detected by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and Coomassie staining of column fractions, pooled and
dialyzed against buffer A, and separated using hydroxyapatite as
described above. Purified YDJ1 or ydj1Q34 was then dialyzed against
buffer B, aliquoted, and snap frozen in liquid N
before
storage at -80 °C.
For overexpression in and purification from E.
coli, a 1.3-kilobase PCR product comprising the open reading frame
of ydj1Q34 was cloned in-frame behind the T7 promoter, into the NcoI and BamHI sites of pET9d (Novagen). The
construct was then transformed into BL21(DE3), which contains the T7
polymerase gene under the control of the lacUV5 promoter
(Studier et al., 1990). The resulting strain overexpressed
ydj1Q34 upon induction with
isopropyl-1-thio--D-galactopyranoside, as roughly 30% of
total protein. Overexpression of wild type YDJ1 in BL21(DE3) was
described previously (Caplan et al., 1992b). General molecular
biology methods followed those in Sambrook et al.(1989).
Nondenaturing gel shift assays were performed to assess the binding
of substrate by Hsp70 as described previously (Cyr et al., 1992). Carboxymethyllactalbumin (CMLA, Sigma) was
iodinated with Na
I (ICN Radiochemicals) using the
IODO-BEAD (Pierce) method according to the manufacturer's
instructions and desalted on a G-25 column preblocked with bovine serum
albumin. Radiolabeled CMLA was incubated with SSA1, YDJ1, ydj1Q34, and
peptides at indicated concentrations, in 40-µl samples containing a
reaction buffer of 50 mM HEPES, pH 7.4, 50 mM NaCl,
10 mM DTT, 1 mM ATP, 2 mM MgCl
,
0.4% bovine serum albumin. After pipetting on ice, the reactions were
incubated at 30 °C for 15 min. Reactions were stopped on ice, and
20 µl of ice-cold reaction buffer in 40% glycerol, 0.01% bromphenol
blue was added to each sample. Thirty µl of each sample was loaded
onto duplicate nondenaturing 7.5-15% acrylamide nondenaturing
gels, which were run on ice at 10 mA/gel. Gels were dried and exposed
to x-ray film.
I-CMLA-SSA1 complexes were quantitated by
densitometry (Molecular Dynamics)
Figure 1:
YDJ1 structure and J-domain peptides. Panel A, amino acids 1-70 of YDJ1 comprise the J-domain,
the presence of which defines it as a DnaJ homologue. This conserved
J-domain is followed by a flexible glycine/phenylalanine-rich region
between positions 80 and 104, four repeats of the motif
CXXCXGXG between positions 143 and 207, and
a region of lower homology, which are also present in DnaJ and some
yeast homologues. Finally, the COOH terminus of YDJ1 is uniquely
farnesylated. Panel B, the J-domain forms four -helices
in solution. Four peptides, p1-20, p21-40, p41-60,
and p61-80, were designed to span the J-domain. Another four
peptides, YHPD, YHPDX
, X
YHPDx
, and p21-40H34Q,
were utilized to assess the role of amino acids 34-36 in YDJ1
function. p21-40H34Q contains the same sequence as p21-40
except for the substitution of glutamine for histidine at position
34.
Figure 2:
Ability
of p21-40 to reduce YDJ1-stimulated Hsp70 ATP hydrolysis. 0.5
µM purified SSA1 or 0.5 µM SSA1 and 1.0
µM YDJ1 was incubated with 0.0, 1.0, 10.0, 20.0, and 50.0
µM p1-20 or p21-40 in the presence of
Mg and [
-
P]ATP. ADP
formation was determined after a 15-min incubation at 30 °C by
spotting a 2-µl aliquot onto polyethyleneimine-cellulose,
chromatographic separation, and quantitation of radioactivity in ADP-
and ATP-containing spots. Results were normalized with respect to total
counts in each sample and adjusted for spontaneous ATP hydrolysis.
Neither p1-20 nor p21-40 stimulated SSA1 ATPase activity.
However, p21-40 reduced ATP hydrolysis in the samples containing
YDJ1, indicating that p21-40 might be competing with YDJ1 for
interaction with SSA1 and that a sequence within amino acids
21-40 of the J-domain mediates this interaction. By contrast, no
competition was observed for p1-20.
To identify candidate sequences
within p21-40 which might mediate interaction with Hsp70, we
considered both sequence conservation and the context within the
predicted protein structure. Although the entire J-domain is quite
highly conserved, the amino acids at positions 34-36 (HPD) are
absolutely conserved between DnaJ and all of the known yeast
homologues. Moreover, this sequence is situated in a loop or hinge
region between the two -helices that were initially predicted
after sequence analysis of DnaJ (Bardwell et al., 1986). These
helices might serve to present a conserved loop sequence for
interaction with other proteins. To test the possibility that this
conserved sequence could interact with Hsp70, three synthetic peptides
from this region were used to compete with YDJ1p. A 4-mer with the
sequence YHPD could not compete off YDJ1-stimulated ATPase activity. We
examined the possibility that sequences within amino acids 21-32
were responsible for the reduction in ATPase stimulation by using the
20-mer pYHPDX
in competition experiments. This
peptide, corresponding to amino acids 33-52, exhibited a weak
ability to compete compared with p21-40. This weak competition
could not be attributed to amino acids 41-52, since p41-60
did not compete. Because amino acids 21-32 were not present on
this peptide and because residues 37-41 are not well conserved,
it seemed plausible that the HPD sequence was responsible for this
competition. It was reasoned that the position of the conserved
sequence within a competing peptide might be important. The 10-mer X
YHPDX
containing the
candidate sequence nestled in the center was slightly better in
competition experiments than YHPDX
( Fig. 3and Table 1). Thus, amino acids 34-36 of the
J-domain, HPD, appear necessary but not sufficient for full stimulation
of Hsp70 ATPase activity.
Figure 3:
HPD peptide competition ATPase assays.
Increasing amounts of peptides YHPD, YHPDX, and X
YHPDX
were added to samples
containing 0.5 µM SSA1 and 1.0 µM YDJ1.
Samples were incubated for 15 min at 30 °C, after which
[
-
P]ADP formation was assessed. An aliquot
from each sample was removed for separation by thin layer
chromatography and quantitation. Results were normalized for total
counts in each sample, and the nonstimulated SSA1 activity was
subtracted. The 4-mer was unable to compete with YDJ1 for interaction
with SSA1. Somewhat longer HPD-containing peptides, however, could
interact. Inclusion of a 100 molar excess of YHPDX
reduced the YDJ1-stimulated SSA1 ATPase activity by 12%.
Likewise, the 10-mer X
YHPDX
reduced the rate of ADP hydrolysis by
30%.
Previous data from this laboratory
demonstrated that YDJ1 is farnesylated (Caplan et al., 1992b)
and that Hsp70 is most likely to be YDJ1's partner
in the yeast cytosol (Cyr and Douglas, 1994). Although farnesylation
increases YDJ1 association with a membrane fraction following heat
shock, this modification is known to mediate specific protein-protein
interactions (Marshall, 1993). To test the possibility that
COOH-terminal farnesylation of YDJ1 confers specificity of interaction
with SSA1, two peptides corresponding to the COOH-terminal sequence
were used in competition experiments. pSASQ contains the COOH-terminal
13 amino acids encoded by the nucleotide sequence, with the exception
that amino acid 406 (the C of the CaaX box) is changed to
serine. This substitution results in a TS phenotype (Caplan et
al., 1992b) and defective transport of polypeptide precursors into
organelles in vivo (Caplan et al., 1992a).
pC-farnesyl corresponds to the farnesylated and proteolytically
processed COOH terminus of YDJ1. Neither peptide showed any detectable
competition of YDJ1-stimulated Hsp70 ATPase activity, even at
peptide:YDJ1 molar ratios of 100:1.
Figure 4:
Peptide
competition gel shift experiments. Nondenaturing gel electrophoresis
and autoradiography were used to evaluate I-CMLA-SSA1
complexes in the presence of J-domain peptides (top panel).
The star (
) marks the position of the CMLA-SSA1
complex. 1.0 µM radioiodinated CMLA was incubated with 0.5
µM purified SSA1 in the presence of Mg-ATP, forming a
I-CMLA-SSA1 complex (lane 1). Inclusion of 1.0
µM YDJ1 stimulates release of CMLA from SSA1 (lane
2). J-domain peptides do not prevent formation of CMLA-SSA1
complex, as there is no significant decrease of complex with increasing
peptide addition (lanes 3 and 5, 7, and 9). When added to samples containing YDJ1 as well, 10
µM p1-20 could not compete off the YDJ1-stimulated
CMLA (lane 4), although 100 µM p1-20 could
do so to some extent (lane 6). By contrast p21-40
appears to have a reproducible stabilizing effect on the complex at
both 10 µM and 100 µM concentrations (lanes 8 and 10). These results were quantitated and
confirmed by densitometry (bottom panel). Numbered lanes shown in the histogram correspond to those lanes scanned on the
autoradiogram above. The amount of
I-CMLA complex present
in the absence of either peptide or YDJ1 (lane 1) was taken as
100% bound.
Figure 5:
Comparison of YDJ1 and ydj1Q34 stimulation
of Hsp70 ATPase. To compare the ability of wild type YDJ1 and the
mutant protein ydj1Q34 to stimulate SSA1, 0.5 µM purified
SSA1 was incubated under assay conditions with either 0.5, 1.0, 2.0, or
5.0 µM ydj1Q34 or the same concentrations of ydj1Q34 and
YDJ1. Samples were incubated for 15 min at 30 °C, after which a
2-µl aliquot was removed. [-
P]ADP and
[
-
P]ATP were separated by thin layer
chromatography and quantitated. Values were normalized with respect to
total counts in each sample. Because the ydj1Q34 preparation contained
a small amount of contaminating ATPase, the values for ADP formation in
control samples containing only the indicated concentrations of ydj1Q34
in reaction buffer were subtracted from each set of experimental
samples. The mutated protein ydj1Q34 lacked the ability to stimulate
Hsp70
even when present in 10-fold molar
excess.
The ability of
ydj1Q34 to stimulate substrate release and to compete with YDJ1 for
substrate release was also examined. Ability to compete without ability
to stimulate would indicate that binding of YDJ1 alone is not
sufficient for the conformational change in SSA1 which results in
peptide substrate release. Fig. 6shows that although a 2:1
molar ratio of YDJ1:SSA1 effects release of bound I-CMLA (lane 2), the same ratio of ydj1Q34 could not stimulate any
release (lane 3). However, this inability does not stem from a
total inability to bind to SSA1p. We observed that a 10-fold excess of
ydj1Q34 could prevent YDJ1 stimulation of CMLA release from
Hsp70
(lane 4).
Figure 6:
ydj1Q34 gel shift experiments. 1.0
µM radiolabeled CMLA and 0.5 µM SSA1 were
incubated under nondenaturing gel shift assay conditions alone and in
the presence of either 1.0 µM YDJ1, 1.0 µM ydj1Q34, or 1.0 µM YDJ1 + 10.0 µM ydj1Q34. After a 15-min incubation at 30 °C, samples were
separated on nondenaturing gels, which were then dried and exposed to
x-ray film (top panel). After developing, the SSA1-CMLA
complexes (as shown alongside the star) were quantitated by
densitometry (bottom panel). Histogram bars correspond to gel lanes of the same number. The amount of I-CMLA-SSA1 complex in lane 1 was taken to be
100% bound. Although ydj1Q34 does not stimulate release of
I-CMLA bound to SSA1 (lane 3), a 10-fold excess
of ydj1Q34 (lane 4) can significantly reduce the amount of
release effected by the wild type YDJ1 protein (lane
2).
These results indicate two
possibilities: either that ydj1Q34 possesses a weaker affinity for
Hsp70 or that ydj1Q34 possesses wild type affinity but
that upon binding the mutant ydj1Q34 protein does not stimulate ATPase
activity and substrate release. To distinguish between these two
possibilities, we used peptide p21-40H34Q in competition
experiments (Fig. 7). p21-40H34Q mimics the amino acid
change in ydj1Q34 and is otherwise identical to p21-40. If
ydj1Q34 affinity for SSA1 remains the same as that of the wild type
YDJ1, then p21-40H34Q and p21-40 (wild type) should compete
off the YDJ1-stimulated ATPase activity of SSA1 equally well. The
addition of 50 µM p21-40 to a reaction mixture
containing 0.5 µM SSA1 and 1.0 µM YDJ1
decreased maximal ATP hydrolysis by 72%; however, the addition of 50
µM p21-40H34Q achieved only a 35% reduction. These
and the data in Fig. 6suggest that ydj1Q34 binds to SSA1 with
less affinity than wild type YDJ1 and that the ydj1Q34-SSA1 interaction
is not transduced into hydrolysis of ATP by SSA1 and release of bound
polypeptide substrate. It appears that the conserved HPD of the
J-domain is important to the binding of YDJ1 to SSA1 and is absolutely
required for YDJ1 stimulation of SSA1 ATPase activity, which is in turn
coupled to polypeptide binding and release.
Figure 7:
pH34Q competition for Hsp70 interaction.
SSA1 at 0.5 µM and YDJ1 at 1.0 µM were
incubated in reaction buffer with 0.0, 10.0, 20.0, and 50.0 µM p21-40 (wild type (WT) sequence) or
p21-40H34Q to determine whether they could reduce ATP hydrolysis
equally well. Samples were incubated for 15 min at 30 °C, after
which 2-µl aliquots were removed and spotted in duplicate onto
polyethyleneimine-cellulose. After separation of
[-
P]ADP and
[
-
P]ATP by thin layer chromatography, spots
were visualized, excised, and counted. Plotted values were normalized
for the number of total counts present in each sample and adjusted for
the amount of nonstimulated SSA1 ATP hydrolysis. Although
p21-40H34Q reduced YDJ1-stimulated ATPase activity to 65% of that
in the absence of peptide, it could not compete off YDJ1 as effectively
as the wild type sequence.
The result that YDJ1 interacts with SSA1 at a site distinct
from that for peptide substrate binding is consistent with a current
model proposing that in addition to stimulating Hsp70 ATPase activity,
J homologues initially bind to some unfolded proteins and nascent
polypeptides and recruit Hsp70 to its target substrates. DnaJ (Hendrick et al., 1993) and the yeast J homologues SIS1 (Zhong and
Arndt, 1993) and YDJ1 associate with polysomes. DnaJ is
known to initially bind and then recruit DnaK for binding to both the
heat shock sigma factor,
32 (Gamer et al., 1992) and
RepA protein (Wickner et al., 1992). Upon recruitment, the
DnaK conformation is altered by interaction with DnaJ such that ATP
hydrolysis is favored over ATP binding. Liberek et al. (1991b)
have demonstrated using partial tryptic digests that DnaK undergoes
different conformational changes in the presence of either ATP or DnaJ.
In addition, wild type DnaJ can either prevent or quickly relax the ATP
associated change in DnaK trypsin susceptibility, whereas the dnaJ259
mutant protein does not (Wall et al., 1994). If a J-protein
acts to recruit Hsp70 to polypeptide substrates, then the J-protein
effect on Hsp70 conformation must be concurrent with or precede binding
of substrate peptide by Hsp70. The substrate binding site of Hsp70
therefore must be available to the peptide and not occupied by the
J-protein.
In this study, the peptide p21-40 competes with YDJ1 for stimulation of SSA1 ATPase activity and CMLA release but does not prevent formation of the CMLA-SSA1 complex. This argues against any model that proposes that substrate release from Hsp70s results from direct displacement by J-proteins. It is possible, however, that J-protein interaction and peptide substrate binding may still be mutually exclusive because of differing conformations of Hsp70 for binding of either. Although p21-40 could not prevent binding of CMLA to SSA1 at concentrations that prevented release of bound CMLA, the 20-mer is unlikely to have had any effect on SSA1 conformation, since it could not alone stimulate SSA1 ATPase activity. Ydj1Q34 was able to interact with SSA1 to prevent YDJ1-stimulated release of CMLA in gel shift experiments; however, it too is unlikely to affect SSA1 conformation since it could not alone stimulate ATPase activity or peptide release. This is analogous to dnaJ259, which was shown to have reduced influence on DnaK conformation relative to wild type DnaJ. The conformational change in Hsp70 protein upon interaction with wild type J-proteins would result in two different populations of Hsp70 molecules, reflecting those that had interacted with J-proteins and those that had not. This may explain why we never observed complete reduction of SSA1 ATPase activity to basal levels in the presence of YDJ1 and a large excess of the competing peptide p21-40.
It is
likely that other regions of the J-domain are also sites of interaction
with Hsp70s. In our study, shorter HPD-containing peptides could not
compete off YDJ1-stimulated Hsp70 ATPase activity, indicating that
other residues must be necessary for YDJ1-Hsp70 binding. It was
surprising that none of the other three J-domain peptides, p1-20,
p41-60, and p61-80, exhibited any measurable effect on the
YDJ1-stimulated increase in SSA1 ATPase activity, despite the presence
of very highly conserved sequences throughout the J-domain spanned by
these peptides. Recent publication of the the NMR structure for amino
acids 2-108 of DnaJ suggests a model that accounts for the
importance of both the HPD sequence in the interhelical hinge region
and the selective pressure for sequence conservation along the rest of
the J-domain. Two groups (Szyperski et al., 1994; Hill et
al., 1995) found independently that in contrast to the predicted
structure of two -helices, the monomeric J-domain possesses four
helices that interact strongly with one another to form a hydrophobic
core. The two longer helices 2 and 3 associate with one another to
present the conserved HPD in the interhelical loop. In this model, the
sequence conservation in the helical regions of the J-domain preserves
the precise interactions that determine the tertiary structure of the
domain, which is necessary for presentation of the HPD sequence. While
this manuscript was in review, several nonconserved positions within
the J-domain were also identified as necessary for binding to Hsp70.
These nonconserved residues serve to determine the specificity of J
homologues for different Hsp70 molecules (Schlenstedt et al.,
1995). Taken together, these data on the roles of conserved and
nonconserved residues within the J-domain suggest that tertiary
conformations are important for the presentation of specific residues
necessary for recognition, binding, and stimulation. The 20-mer
peptides p1-20, 41-60, and 61-80 are too short to
attain these conformations. By contrast, p21-40 largely
corresponds to a loop region. Because the J-domain is monomeric in
solution, p21-40 cannot prevent YDJ1 stimulation of Hsp70 by
disruption of the YDJ1 dimer.
That several nonconserved residues
have been identified as necessary for J homologue binding to specific
cognate Hsp70s suggests that the absolutely conserved HPD sequence is
specifically required for stimulation of ATPase activity. This
conclusion is supported by our demonstration that the ydj1Q34 mutant
protein can prevent YDJ1-stimulated release of bound CMLA by
Hsp70, despite its total lack of ability to stimulate
either ATPase activity or substrate release on its own. The mutant
protein was able to prevent CMLA release at lower ratios relative to
YDJ1 than those required for competition by p21-40 peptide ( Fig. 4and Fig. 6and data not shown), thus suggesting
that the full-length protein is capable of binding but not stimulating
Hsp70. This ability of ydj1Q34 to bind Hsp70 nonproductively, as well
as its solubility and purification characteristics, which are identical
to those of the wild type YDJ1 protein, confirms that the mutant is not
dysfunctional because of any misfolded structure.
It is noteworthy
that amino acids 22-29 in the DnaJ from Clostridium
acetobutylicum, KKAFRKLA (Behrens et al., 1993), are
similar to a sequence of Raf kinase, RKTFLKLA, which may interact with
-phosphate residues of ATP. (
)Amino acids 23-30
of YDJ1, KKAYEKCA (Caplan and Douglas, 1991) and 22-29 of E.
coli DnaJ, RKAYKRLA (Bardwell et al., 1986; Ohki et
al. 1986) are less similar; however, it is tantalizing to
speculate that this sequence present in helix 2 of the J-domain and
represented in the competing peptide p21-40 may also have a role
in regulating Hsp70 activity. If this is the case, then J-proteins
would interact with Hsp70s at or near their catalytic ATPase domain.
It is now confirmed that the J-domain of DnaJ and a eukaryotic homologue, YDJ1, are necessary to effect a conformational change in Hsp70 leading to increased ATPase activity and peptide substrate release. What then, is the function of the remainder of the YDJ1 molecule, which shares other conserved domains with DnaJ? Szypersky et al.(1994) have shown that the glycine/phenylalanine-rich stretch immediately COOH-terminal to the J-domain of DnaJ is flexible in solution, possibly to allow proper orientation of the J-domain for interaction with DnaK. The zinc finger domain, containing four repeats of the motif CXXCXGXG, has an unknown function. Although DnaJ does function in DNA replication, there has been no demonstration of DNA binding capabilities. Moreover, this motif is conserved in the cytosolic protein YDJ1. Caplan et al. (1992a) have shown genetically that YDJ1 functions as a dimer; possibly this domain serves as a dimerization site. YDJ1 associates with membranes upon heat shock in a farnesylation-dependent manner (Caplan et al., 1992b); however, this farnesylation appears to be a signal for translocation upon heat shock and may not confer intrinsic affinity for membranes.
The zinc fingers might be analogous to the ``zinc butterflies'' described for protein kinase C and Raf kinase, which bind phospholipids and phorbol esters (Quest et al., 1992; Ghosh et al., 1994; Ghosh and Bell, 1994; Lehel et al., 1995). This region of protein kinase C has been suggested as a site for interaction with 14-3-3 proteins (Robinson et al., 1994). Alternatively, this domain may provide a structural motif for the recruitment of Hsp70. Finally, the COOH-terminal portion of YDJ1 is far less conserved between DnaJ and the other J homologues and ends with a farnesyl modification unique to YDJ1 among yeast homologues. Two plant dnaJ homologues, LDJ1 in Allium porrum (Bessoule, 1993) and ANJ1 in Atriplex numularia (Zhu et al., 1993) and one human homologue HDJ2 (Chellaiah et al., 1993) also contain a COOH-terminal farnesylation signal. ANJ1 in A. numularia has been show to be farnesylated and, like YDJ1, is necessary for translocation to membranes and survival at elevated temperatures. It is likely that the COOH-terminal region of YDJ1 is specialized for YDJ1's role as a cytoplasmic chaperone that also associates with organellar membranes to deliver protein substrates.