Purification of a 38-kDa Protein from Rabbit Reticulocyte
Lysate Which Promotes Protein Renaturation by Heat Shock Protein 70 and Its Identification as
-Aminolevulinic Acid Dehydratase and
as a Putative DnaJ Protein*
Martin
Gross
,
Suzanne
Hessefort, and
Annette
Olin
From the Department of Pathology, The University of Chicago,
Chicago, Illinois 60637
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ABSTRACT |
We reported recently that a
rabbit reticulocyte 66-kDa protein (termed RF-hsp 70 by our laboratory
and p60 and hop by others) functions as a hsp 70 recycling protein and
markedly enhances the renaturation of luciferase by hsp 70 (Gross, M.,
and Hessefort, S. (1996) J. Biol. Chem. 271, 16833-16841). In this report, we confirm that the ability of RF-hsp 70 to promote the conversion of hsp 70·ADP to hsp 70·ATP, thus
enhancing the protein folding activity of hsp 70, is caused by the
purified 66-kDa protein and not by a trace DnaJ/hsp 40 protein
contaminant. To determine the relationship between RF-hsp 70 and the
DnaJ/hsp 40 heat shock protein family, which also enhances protein
renaturation by hsp 70, we purified a 38-kDa protein from rabbit
reticulocyte lysate based upon its ability to stimulate renaturation of
luciferase by hsp 70. Partial amino acid sequencing of this 38-kDa
protein has indicated, unexpectedly, that it is the enzyme
-aminolevulinic acid dehydratase (ALA-D) and that it does not
contain detectable sequences corresponding to the DnaJ/hsp 40 protein
family. In addition, immunoblot analysis with a polyclonal antibody
made to HeLa cell hsp 40 (from StressGen) confirms that our purified ALA-D contains no hsp 40, although hsp 40 is present in relatively crude rabbit reticulocyte protein fractions. Rabbit reticulocyte ALA-D
is about as active in converting
-aminolevulinic acid to porphobilinogen and as Zn2+-dependent as ALA-D
purified from other sources. Rabbit reticulocyte ALA-D stimulates the
renaturation of luciferase by hsp 70 up to 10-fold at concentrations
that are the same as or less than that of hsp 70, and it has no
renaturation activity in the absence of hsp 70. The renaturation effect
of ALA-D is additive with that of RF-hsp 70 at limiting or saturating
concentrations of each, and, unlike RF-hsp 70, ALA-D does not promote
the dissociation of hsp 70·ADP in the presence of ATP. The
renaturation-enhancing effect of ALA-D may be caused by a region near
its carboxyl terminus which has sequence homology to the highly
conserved domain of the DnaJ protein family, which is similar to the
sequence homology between this domain and a carboxyl-terminal region in
auxilin, a DnaJ-like protein that requires this region for its hsp
70-dependent function (Ungewickell, E., Ungewickell, H.,
Holstein, S. E. H., Lindner, R., Prasad, K., Barouch, W., Martin, B.,
Greene, L. E., and Eisenberg, E. (1995) Nature 378, 632-635).
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INTRODUCTION |
We reported recently the purification of a 66-kDa protein from
rabbit reticulocyte lysate which is found associated with hsp 701; we termed it RF-hsp 70 because it promotes the recycling of hsp 70 by stimulating the
dissociation of ADP from, and the binding of ATP to, hsp 70 (1).
Consistent with a recycling function, RF-hsp 70 increases by 10-fold
the rate of renaturation of heat-denatured luciferase by hsp 70 (1).
Extensive amino acid sequence analysis of RF-hsp 70 demonstrated (2)
that it is structurally homologous to human IEF SSP 3521 (3), mouse
extendin (4), and chicken p60 (5), suggesting that these proteins may
also function to recycle hsp 70. However, when recombinantly expressed
(in bacteria) IEF SSP 3521 was tested, it failed to enhance the
renaturation of luciferase (6) or
-galactosidase (7) by hsp 70, in
contrast to the activity of RF-hsp 70. Further testing of the
recombinant human homolog of RF-hsp 70 (termed hop) by Johnson et
al. (8) showed that although it does bind to hsp 70 and to hsp 90 and does stimulate luciferase renaturation in the presence of hsp 70 and YDJ-1 (a yeast DnaJ/hsp 40 homolog) or hsp 70, YDJ-1, and hsp 90, it is inactive with hsp 70 alone, in contrast to our previous findings
(1). Furthermore, the recombinant hop failed to stimulate the ATPase
activity of hsp 70, the dissociation of hsp 70·ADP, and the binding
of ATP to hsp 70 (8), all in contrast to our report (1). In addition,
several recent reports have demonstrated that members of the DnaJ/hsp
40 protein family, YDJ-1 and Hdj-1, do promote, respectively, the
renaturation of luciferase (9) and
-galactosidase (7, 10) by hsp 70, similar to the effect of RF-hsp 70. Because of these contrasting
findings and the finding that trace amounts of DnaJ/hsp 40 protein can
contaminate other proteins involved in the renaturation process and
influence their activity (9, 11), we tested and will show that there is
no detectable, silver-stained protein migrating further than the 66-kDa
protein on SDS-polyacrylamide gel electrophoresis, even when a 10-fold
excess of purified RF-hsp 70 is analyzed, and that RF-hsp 70 contains
no hsp 40 as determined by immunoblot analysis with antibody
to hsp 40.
To determine the relationship between the effects of RF-hsp 70 and the
DnaJ protein family, we purified a protein from rabbit reticulocyte
lysate which stimulates the hsp 70-mediated renaturation of denatured
luciferase by a mechanism that is distinct from that of RF-hsp 70. This
protein migrates as a 38-kDa band on SDS-polyacrylamide gel
electrophoresis, the size characteristic of the DnaJ protein family.
Partial amino acid sequence analysis of this protein has demonstrated,
however, that it is the enzyme
-aminolevulinic acid dehydratase
(ALA-D), and immunoblot analysis has confirmed that it is free of any
hsp 40. In addition, enzymatic assay has demonstrated that the purified
38-kDa protein converts
-aminolevulinic acid (ALA) to
porphobilinogen (PBG). We will show that the ability of this protein to
enhance renaturation of luciferase by hsp 70 may be caused by a region
near its carboxyl terminus which has considerable sequence homology to
the highly conserved J domain characteristic of the DnaJ protein
family. In this regard, the protein folding enhancement we have
observed with ALA-D may be similar to the role that auxilin is believed
to play in the hsp 70-mediated uncoating of reconstituted clathrin
baskets (12, 13). Auxilin action is believed to be mediated by a region
near its carboxyl terminus which shows the same degree of homology to
the DnaJ consensus sequence (12, 13) which we have found in ALA-D.
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EXPERIMENTAL PROCEDURES |
Purification of Protein Components--
The purification of hsp
70 from rabbit reticulocyte lysate was as described previously (14),
and its purity is demonstrated in Fig. 1B (lane
9). Its migration upon SDS-polyacrylamide gel electrophoresis and
sequencing of a 35-amino acid polypeptide containing its carboxyl
terminus, obtained by limited chymotrypsin digestion, indicate that
this hsp 70 is the constitutively expressed form or hsc 73 (1). Unless
otherwise indicated, this was the hsp 70 preparation used in all
experiments. The purification of hsp 90 and RF-hsp 70 from rabbit
reticulocyte lysate was as described (1), except that the final
purification step (step 6) for RF-hsp 70 employed chromatography on a
Superdex 200 HR 10/30 column (1.0 × 30 cm; Amersham Pharmacia
Biotech). Step 5 RF-hsp 70 (0.50 ml and up to 4 mg/ml protein) was
applied, and the column was equilibrated and eluted with 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM dithiothreitol, and 0.1 mM EDTA at a flow
rate of 0.50 ml/min using the Amersham Pharmacia Biotech fast protein
liquid chromatography system. The absorbance at 280 nm was monitored,
and 0.50-ml fractions were collected. This step 6 RF-hsp 70 appears
homogeneous even when a large excess (2 µg) is subjected to gel
electrophoresis and silver staining (see Fig. 1A). We have
found recently that chromatography on Mono Q, used previously to
prepare step 6 RF-hsp 70 (1), may result in a major loss of activity.
The initial purification of a 38-kDa protein from rabbit reticulocyte
lysate, involving preparation of the postribosomal supernatant (step
1), precipitation at pH 5.2 (step 2), 0-40% ammonium sulfate precipitation (step 3), and then chromatography on DEAE-cellulose (step
4), was as described for the initial purification of ProHCR (15). Up to
10 mg of step 4 protein was then applied to a 0.5 × 5.0-cm (1.0 ml) Mono Q column (Amersham Pharmacia Biotech) equilibrated in 20 mM Tris-HCl, pH 7.5, 100 mM KCl, and 0.1 mM EDTA. After washing with 3.0 ml of equilibration buffer,
the column was developed at a flow rate of 1.0 ml/min and brought to 20 mM Tris-HCl, pH 7.5, 240 mM KCl, 0.1 mM EDTA in 3.0 min and then to 20 mM Tris-HCl, pH 7.5, 500 mM KCl, 0.1 mM EDTA over an
additional 14.0-min period. The 38-kDa protein, eluting at about 380 mM KCl, was pooled from multiple runs and concentrated by
ultrafiltration to about 1.0 mg/ml (Amicon; YM-5 membrane) and made
10% (v/v) with glycerol to give step 5 protein. This preparation (0.50 ml) was then chromatographed on Superdex 200 exactly as described above
for preparing step 6 RF-hsp 70. The 38-kDa protein (step 6) was pooled
from multiple runs, concentrated by ultrafiltration to about 1.5 mg/ml,
made 10% (v/v) with glycerol, and stored in liquid nitrogen. It
appears homogeneous when analyzed by SDS-polyacrylamide gel
electrophoresis and silver staining (see Fig. 1B).
Renaturation of Luciferase--
The ability of specific proteins
to promote the renaturation of heat-denatured luciferase was determined
as described previously (1, 6). In brief, firefly luciferase was
diluted to about 0.5 nM, duplicate aliquots were removed
for determination of the initial activity, and the remainder was heated
at 40 °C for 20 min, reducing luciferase activity to about 6% of
its initial activity. When samples were renatured for a single time
period, renaturation reactions, in duplicate, contained 2.5 µl of
heated luciferase, 0.50 mM ATP, 15 mM creatine
phosphate, 45 units/ml creatine phosphokinase, and the protein
additions indicated in the figure and table legends in a final volume
of 25 µl of 10 mM Tris-HCl, pH 7.5, 100 mM
KCl, 3 mM MgCl2, 4 mM
dithiothreitol, and 0.1 mM EDTA. When renaturation was
followed with time, samples were similar but scaled up to 110 µl.
Incubation was at 32 °C and for the indicated times. Luciferase activity was measured in 25-µl samples by mixing with 100 µl of 25 mM Tricine, pH 7.8, 5 mM MgCl2, 1.7 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM D-luciferin, 0.25 mM coenzyme A,
and 0.5 mM ATP and counting immediately in a TM Analytic
model 6895 liquid scintillation spectrometer (1). Results are expressed
as the percentage of the initial activity of unheated luciferase.
Assay for the Conversion of ALA to PBG by ALA-D--
Partially
purified or pure preparations of the 38-kDa protein were assayed for
ALA-D activity under conditions that were similar to those used for
assessing the effect of the 38-kDa protein on the renaturation of
luciferase described above. Duplicate samples contained up to 60 µg/ml 38-kDa protein and 2 mM ALA in a final volume of
150 µl of 30 mM Tris-HCl, pH 7.5, 100 mM KCl,
3 mM MgCl2, 5 mM dithiothreitol,
0.1 mM EDTA, and 5.0 µM ZnCl2.
Incubation was at 37 °C, and 50-µl aliquots were removed at 7.5, 15, and 30 min to determine the rate of formation of PBG. In one
experiment (see Fig. 4), the concentration of added ZnCl2
varied, and in another (see Fig. 5), samples were incubated in 5 mM dithiothreitol and either 50 mM Tris-HCl or
sodium phosphate at the indicated pH values. PBG was determined as
described by Sassa (16) by mixing 50-µl aliquots with 125 µl of 6%
(w/v) trichloroacetic acid and 0.1 M HgCl2,
centrifugation, and removal and mixing of 150 µl of each supernatant
with 150 µl of modified Ehrlich's reagent (16). After maximal color
development (10-15 min at room temperature), the absorbance at 553 nm
was determined and converted to µmol of PBG formed/h using a molar
absorption coefficient of 6.1 × 104 (16).
Partial Amino Acid Sequencing of the 38-kDa
Protein--
Sequencing was performed in an Applied Biosystems 473A
sequencer equipped with an on-line ATZ to phenylthiohydantoin
autoconverter, a gradient-programmable microbore chromatography system,
and a Macintosh-based 610A data system to identify and quantitate the amino acid residues. Amino-terminal sequencing of the intact, step 6, 38-kDa protein was performed (see Fig. 2). In addition, 90 µg of this
protein was digested with 7.5 µg of trypsin for 9.2 h at
34 °C, and the tryptic peptides were isolated by reverse phase
chromatography on an Aquapore RP-300 column as described previously
(2).
Gel Electrophoresis--
Electrophoresis of protein samples on
7% polyacrylamide-SDS slab gels (17) followed by silver staining (18)
has been described previously.
Materials--
Reagents for the renaturation and assay of
firefly luciferase were all from Sigma, except that dithiothreitol was
obtained from Calbiochem. All protein standards and ALA were purchased from Sigma, and [
-32P]ATP was purchased from ICN.
Reagents for gel electrophoresis were from Bio-Rad, and electrophoresis
equipment was from Hoefer.
 |
RESULTS |
Purification of a Rabbit Reticulocyte Protein, Distinct from RF-hsp
70, Which Stimulates the Renaturation of Luciferase by Hsp 70--
To
verify that the activity of RF-hsp 70 is attributable to its 66-kDa
component, which is homologous to human IEF SSP 3521 (3), mouse
extendin (4), and chicken p60 (5), and not to a possible DnaJ protein
contaminant, we subjected a large excess of several step 6 RF-hsp 70 samples to SDS-polyacrylamide gel electrophoresis and silver staining.
The results (Fig. 1A)
demonstrate that these preparations contain no protein migrating faster
than the 66-kDa band, indicating that they are devoid of a DnaJ protein member, which would migrate at about 40 kDa. In addition, immunoblot analysis of an excess of steps 5 and 6 RF-hsp 70, using a polyclonal antibody made against HeLa cell hsp 40 (see Fig. 3, lanes 10 and 8, respectively), confirms that these preparations
are devoid of any hsp 40.

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Fig. 1.
Gel electrophoretic analysis of an excess of
purified RF-hsp 70 and the purification steps of a rabbit reticulocyte
38-kDa protein. Protein samples were subjected to
SDS-polyacrylamide gel electrophoresis and then silver staining as
described under "Experimental Procedures." Panel A:
lanes 1-3, 2, 1.5, and 2 µg of three different
preparations of purified (step 6) RF-hsp 70. Panel B:
lane 1, 200 µg of step 1, 38-kDa protein
(postribosomal supernatant); lane 2, 30 µg of step 2, 38-kDa protein; lane 3, 28 µg of step 3, 38-kDa protein
(0-40% ammonium sulfate fraction from step 2); lane 4, 19 µg of 40-80% ammonium sulfate fraction from step 2, 38-kDa protein;
lane 5, 3.2 µg of step 4, 38-kDa protein; lane
6, 0.3 µg of step 5, 38-kDa protein; lane 7, 0.25 µg of step 6, 38-kDa protein; lane 8, 0.2 µg of purified
(step 6) RF-hsp 70; lane 9, 0.2 µg of purified, rabbit
reticulocyte hsp 70. The arrows on the left
indicate the migration positions of hsp 70 (73 kDa), RF-hsp 70 (66 kDa), and the 38-kDa protein; the arrows on the
right mark the migration positions of protein standards that
were run in a parallel lane. Electrophoresis was at 44 V (3 V/cm) for
15 h in panel A and 20 h in panel
B.
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To investigate the relationship between the action of RF-hsp 70 and the
DnaJ protein family, we attempted to purify the corresponding DnaJ
protein from rabbit reticulocyte lysate. Using the stimulation of the
renaturation of heat-denatured luciferase by hsp 70 as our assay, we
purified a relatively abundant protein that migrates as a 38-kDa band
on a denaturing gel, a size that is consistent with a DnaJ protein
family member. The purification of this protein, based upon its ability
to enhance hsp 70-mediated renaturation of luciferase, is shown in
Table I, under "Luciferase
renaturation," and gel analysis of the protein at different steps in
the purification is shown in Fig. 1B. The results indicate
that this 38-kDa protein is purified to near homogeneity at step 5 (Mono Q) and to apparent homogeneity at step 6 (Superdex 200), as seen
in Fig. 1B, lanes 6 and 7,
respectively. For comparison, purified RF-hsp 70 and hsp 70 have been
run in lanes 8 and 9, respectively (Fig.
1B). We chose to purify the 38-kDa protein from the pH 5.2 precipitate and 0-40% ammonium sulfate fractions (Fig. 1B,
lanes 2 and 3, respectively) because the pH 5.2 soluble fraction (not shown) and the 40-80% ammonium sulfate fraction
(Fig. 1B, lane 4) have virtually no 38-kDa
protein. In contrast, RF-hsp 70 is found almost entirely in the
40-80% ammonium sulfate fraction, which was used for its purification
(1). The results in Table I, "Luciferase renaturation," indicate
that the 38-kDa protein, based upon its ability to enhance the
renaturation of luciferase by hsp 70, has been purified 29-fold from
the ammonium sulfate fraction (step 3) with a yield of 12%. We are not
able to determine the activity of the 38-kDa protein in steps 1 and 2 because they also contain RF-hsp 70.
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Table I
Purification of a rabbit reticulocyte 38-kDa protein (ALA-D)
A 38-kDa protein (ALA-D) was purified as described under
"Experimental Procedures," and its activity was assessed by the
stimulation of the renaturation of luciferase (left) or the enzymatic
conversion of ALA to PBG (right). One unit of renaturation activity is
defined as the amount required to renature luciferase to 25% of its
initial activity when incubated for 60 min under renaturation
conditions and in the presence of 1.0 µM hsp 70 in a
final volume of 25 µl. One unit of enzymatic activity is defined as
the amount required to produce 1 µmol of PBG/h at 37 °C and pH
7.5. Both assays are described under "Experimental Procedures."
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The 38-kDa Protein Is ALA-D--
We had presumed that the 38-kDa
protein may be a rabbit reticulocyte lysate DnaJ protein (hsp 40)
member because of its size, relative abundance (an estimated
concentration of 0.1 mg/ml as indicated below), and ability to enhance
luciferase renaturation by hsp 70. However, when we subjected the
purified 38-kDa protein to NH2-terminal sequence analysis,
we obtained a single amino acid sequence (20 residues) that corresponds
to the protein ALA-D (see Fig. 2), the
enzyme (EC 4.2.1.24) that catalyzes the synthesis of PBG from two
molecules of ALA. The sequence of the NH2-terminal 20 amino
acid residues of the rabbit reticulocyte 38-kDa protein is identical to
the translated cDNA sequence of human ALA-D at all but the 18th
residue, where serine replaces alanine, as is the case with mouse
ALA-D. Because this finding was unexpected, we subjected the purified
38-kDa protein to trypsin digestion, peptide separation (by reverse
phase chromatography), and additional amino acid sequence analysis to
determine whether the 38-kDa preparation might also contain a known
DnaJ protein, whose size is identical to that of ALA-D but whose amino
terminus is blocked. The sequences of three of these tryptic peptides
corresponded to residues 18-39, 61-74, and 91-118 in ALA-D (Fig. 2),
confirming that the purified 38-kDa component is ALA-D and, apparently,
only ALA-D. Sequencing of 81 of a total of 330 residues demonstrates
that the rabbit reticulocyte protein has 88% identity to human and
mouse ALA-D (Fig. 2).

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Fig. 2.
Partial amino acid sequences of the 38-kDa
protein are virtually identical to the translated sequences of the
cDNA of human, rat, and mouse ALA-D. The amino acid sequence
of the first 150 residues of human ALA-D, determined from the cDNA
as reported by Ishida et al. (46), is shown on each
top line. Under this are aligned the corresponding sequences
of rat (47) and mouse (48) ALA-D, as determined from the respective
cDNA, and then partial sequences derived from
NH2-terminal sequencing of the intact, purified 38-kDa
protein and three major polypeptides isolated from this rabbit
reticulocyte protein after trypsinization and reverse phase
chromatography as described under "Experimental Procedures." The
single-letter code for the amino acids has been used, and an
asterisk denotes a residue that is identical to that of
human ALA-D. Only the amino-terminal 45% of this 330-amino acid
protein is shown because all rabbit 38-kDa protein sequences determined
were contained within this part of the protein.
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Careful examination of the sequencing analyses of the 38-kDa protein
revealed no detectable polypeptides corresponding to the DnaJ/hsp 40 protein family, although we did detect small amounts of two
polypeptides derived from the added trypsin. Nevertheless, other
laboratories have found that small amounts of the DnaJ/hsp 40 protein
family may contaminate preparations of other proteins that promote
protein folding and renaturation and contribute to their effect (9,
11). Therefore, we used polyclonal antibody, raised to HeLa cell hsp 40 (19) and obtained from StressGen, and immunoblot analysis (20) to try
to verify that the renaturation effect of the purified 38-kDa protein
(Table I "Luciferase renaturation") is caused by ALA-D and not
contaminating hsp 40. This analysis (Fig.
3) demonstrated that relatively crude
fractions (steps 1-3, 38 kDa) do contain hsp 40. However, this hsp 40 is almost completely removed in the preparation of step 4, 38 kDa, and
it is totally absent in steps 5 and 6, 38 kDa, demonstrating that the
renaturation effect of the purified 38-kDa protein is not caused by
contaminating hsp 40 and confirming that it is the result of ALA-D
itself. Comparison of the immunoblot (Fig. 3) with a silver-stained gel
run in parallel showed that ALA-D migrates slightly further than hsp
40, consistent with the fact that ALA-D is 330 amino acid residues in
length, whereas hsp 40 is 340 (21).

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Fig. 3.
Immunoblot analysis of the purification steps
of ALA-D and purified RF-hsp 70 and hsp 70 with antibody to hsp
40. The exact same protein fractions applied to lanes
1-7 in Fig. 1B, but with 3-fold more protein, were
applied to lanes 1-7, respectively. Lanes 8-10
received 0.75 µg of pure (step 6) RF-hsp 70 (lane 8), 0.75 µg of pure rabbit reticulocyte hsp 70 (lane 9), and step 5 RF-hsp 70, containing 0.75 µg of the 66-kDa protein (lane
10). After electrophoresis as indicated in Fig. 1B,
proteins were transferred to nitrocellulose and blotted against rabbit
antiserum (1:2,000) to HeLa cell hsp 40 (19) from StressGen and then to
peroxidase-conjugated, goat anti-rabbit IgG antibody (1:500) from ICN
as described previously (20). The arrows on the
left indicate the migration positions of molecular mass
standards and purified, rabbit reticulocyte ALA-D, which were
electrophoresed in parallel on a second gel and identified by silver
staining. The bottom half of the blot is shown.
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Although we have shown that it is rabbit reticulocyte ALA-D that
promotes the renaturation of luciferase by hsp 70 (Table I), an
activity that is characterized in greater detail below, it was
important to determine whether the same protein is also enzymatically
active. The results in Table I indicate that the same fractions that
promote renaturation of luciferase by hsp 70 also convert ALA to PBG,
demonstrating that both activities are associated with the same
protein, ALA-D. The purification and yield of the enzymatic activity
are somewhat greater than those of the renaturation activity (Table I,
"ALA conversion to PBG" versus "Luciferase
renaturation"), and there are probably two reasons for this. The
apparent activation of enzymatic activity at steps 2 and 3 is probably
caused by the removal of uroporphyrinogen I synthetase, which would
reduce the PBG product in assays with cruder fractions. In addition,
there is a significant loss of renaturation activity per 38-kDa protein
in going from steps 3-6 (Table I, "Luciferase renaturation," and
Fig. 1B), whereas enzymatic activity per 38-kDa protein
remains relatively constant for steps 3-6 (Table I, "ALA conversion
to PBG," and Fig. 1B). Because the renaturation activity
copurifies only with the 38-kDa (and no other) protein in steps 3-6
(Fig. 1B), we believe that this, but not the enzymatic,
activity may be partially stabilized by one or more other proteins.
Alternatively, there may be an additional protein in cruder fractions
which also stimulates renaturation of luciferase. In support of the
former possibility, we find that purified (steps 5 and 6) ALA-D
produces the same maximal stimulation of luciferase renaturation as
does step 3 protein, but approximately 1.7-fold more 38-kDa protein is
required (data not shown). The actual purification and yield of ALA-D
are probably appreciably less than the 1980-fold and 25%,
respectively, shown in Table I, "ALA conversion to PBG," as
explained above. Taking this into account, we estimate that the
concentration of ALA-D in rabbit reticulocyte lysate is on the order of
0.1 mg/ml, similar to our estimate of RF-hsp 70 in reticulocyte lysate
(1).
Prior studies have shown that the enzymatic activity of ALA-D is
dependent upon Zn2+ (22-25), and we find that rabbit
reticulocyte ALA-D is also Zn2+-dependent. The
rate of conversion of ALA to PBG by purified reticulocyte ALA-D was
measured in the absence or presence of increasing concentrations of
ZnCl2 (Fig. 4). Enzymatic
activity in the absence of added Zn2+ was initially very
slow but increased progressively with time. We believe that this slow
activation may be caused by trace Zn2+ present in the
reagents. The initial rate of reaction increased steadily with an
increasing concentration of added ZnCl2 from 0.5 to 4 µM with little further activation at 8 µM
ZnCl2 (Fig. 4). In contrast, the rate of reaction at 10-20
min was maximal and quite similar in samples with added
ZnCl2 of 1 µM or more. Our interpretation is
that 1 µM Zn2+ is just sufficient to activate
ALA-D fully, but there is a 5-10-min delay for complete binding and
activation to occur. This lag is largely overcome by adding a more than
saturating concentration of Zn2+ (4 µM or
greater). It is noteworthy that 1 µM Zn2+,
which is just saturating, equals the concentration of the 38-kDa protein in the assay samples, consistent with a stoichiometry of up to
1 g atom of Zn2+ bound/mol of 38-kDa protein if all
added Zn2+ becomes protein-bound. Bevan et al.
(24) concluded that octameric ALA-D from bovine liver contains 4 g
atoms of Zn2+/mol based upon measurements of enzyme
activity and metal binding. Our finding of a 10-fold stimulation of the
initial enzymatic rate of ALA-D by 4-8 µM
ZnCl2 (Fig. 4) is also in good agreement with a similar
dependence of activity upon Zn2+ made with bovine liver
ALA-D (24).

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Fig. 4.
Dependence of ALA-D enzymatic activity upon
Zn2+. The rate of formation of PBG from ALA
in the presence of 40 µg/ml of purified (step 6) ALA-D was determined
as indicated under "Experimental Procedures." Samples contained a
final concentration of 0.0 ( ), 0.50 ( ), 1.0 ( ), 2.0 ( ), 4.0 ( ), and 8.0 (×) µM ZnCl2.
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Native ALA-D occurs as an octameric enzyme with a molecular weight,
based upon density gradient sedimentation or gel filtration analysis,
of 285 kDa (bovine liver) (26), 250 kDa (Rhodobacter spheroides) (27), or 252 kDa (human erythrocyte) (23).
Chromatography of rabbit reticulocyte ALA-D on a calibrated Superdex
200 column (data not shown) indicates an apparent native molecular mass
of about 275 kDa, very similar to that of the other reported ALA-D species.
Our initial assays of the enzymatic activity of ALA-D (Table I) were
performed with Tris-HCl buffer at pH 7.5 to match the pH and buffer
used to measure the renaturation of luciferase by hsp 70 and its
enhancement by ALA-D and RF-hsp 70 (examined in detail below). We also
measured ALA-D enzymatic activity in sodium phosphate buffer at pH 6.5, the approximate pH optimum reported for crude rat liver ALA-D (16) or
ALA-D purified from bovine liver (28) or human erythrocytes (23), to
permit comparison of the specific activity of the purified rabbit
reticulocyte enzyme with that of ALA-D from these other sources.
Unexpectedly, we found that rabbit reticulocyte ALA-D has optimal
enzyme activity at pH 7.5-8.5, whereas it has only about 40% and 10%
of optimal activity at pH 6.5 and 6.0, respectively (Fig.
5). When the pH dependence of enzyme
activity was tested with Tris-HCl in place of sodium phosphate, the pH
optimum and level of enzyme activity were very similar (Fig. 5),
indicating that our result is not peculiar to a specific buffer. The
difference in pH optimum observed is more likely caused by a difference
in species than in tissue of origin because the pH optima of human
erythroid and rat and bovine liver enzymes are similar, whereas that of
rabbit erythroid ALA-D differs. It is of interest that the pH optimum
for rabbit reticulocyte ALA-D is rather similar to that of ALA-D
isolated from R. spheroides (27). When comparison is made at
the respective optimal pH, the specific activity of purified rabbit
reticulocyte ALA-D of 11.5 units/mg compares fairly well with 18 units/mg for the purified human erythrocyte enzyme (23) and 23 units/mg
for bovine liver ALA-D (24).

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Fig. 5.
Effect of pH on the enzymatic activity of
ALA-D. The rate of formation of PBG from ALA by 40 µg/ml step 6 ALA-D was determined at the indicated pH values in samples buffered
with 50 mM sodium phosphate (closed circles) or
Tris-HCl (open circles). All samples contained a final
concentration of 5.0 µM ZnCl2. Aliquots were
removed at 7.5, 15, and 30 min to determine the rate of reaction.
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Characterization of the Renaturation Effect of Rabbit Reticulocyte
ALA-D--
The effects of limiting and saturating concentrations of
purified rabbit reticulocyte ALA-D and RF-hsp 70, added separately and
together, on the renaturation of heat-denatured luciferase by hsp 70 are shown in Fig. 6. In this and other
such experiments, heat treatment reduced luciferase activity to about
6% of the initial activity, and reincubation of luciferase by itself
resulted in virtually no renaturation (Figs. 6 and
7). The concentration of added hsp 70 was
1.0 µM, which we (1) and Schumacher et al. (6)
previously found was just saturating in this renaturation assay.
Incubation was for 60 min, the time where renaturation approaches a
maximum (see Fig. 7). The results (Fig. 6) show that little
renaturation occurs with hsp 70 alone because luciferase activity
increased from 8.5% to only 10.5% of its initial activity before heat
denaturation. In contrast, ALA-D produced a dose-dependent increase in the renaturation of luciferase in the presence of hsp 70 (and absence of RF-hsp 70) which reached a maximum of just more than
30% of the initial luciferase activity. Although the maximal effect of
ALA-D is much less than complete (100%) renaturation, it does
represent a more than 10-fold increase in renaturation over that
produced by incubation with hsp 70 alone. The renaturation effect of
ALA-D is completely dependent upon the presence of hsp 70 because ALA-D
has no effect when hsp 70 is not added (Fig. 6 and Table
II). Thus, as is the case with RF-hsp 70 (1), ALA-D appears to promote protein renaturation by enhancing the
folding or chaperonin activity of hsp 70. The action of ALA-D is not a nonspecific effect of adding more protein because the same amount of
bovine serum albumin or glutathione S-transferase produces no stimulation of luciferase renaturation (Table II). The concentration of ALA-D which is just saturating, 60 µg/ml (Fig. 6), corresponds to
1.6 µM relative to the 38-kDa monomer or 0.2 µM for the octamer, indicating that at saturation, ALA-D
enhances the renaturation activity of hsp 70 at a molar concentration
that is about equal to or less than that of hsp 70.

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Fig. 6.
Effect of ALA-D and RF-hsp 70 on the
renaturation of heat-denatured luciferase by hsp 70. Renaturation
reactions (25 µl), prepared and incubated as indicated under
"Experimental Procedures," contained the indicated concentrations
of purified ALA-D and 0.0 (×, ), 0.14 ( ), 0.25 ( ), 0.50 ( ), 1.0 ( ), or 1.5 (+) µM RF-hsp 70. All samples
also contained 1.0 µM hsp 70 except those depicted with
an ×, which received no added hsp 70. Luciferase activity was measured
after a 60-min incubation as described under "Experimental
Procedures" and is expressed as a percentage of the activity in
luciferase samples that were not heat-denatured. Samples of
heat-denatured luciferase that were not reincubated had 6.6% of the
initial activity. The results are a composite of four separate
experiments.
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Fig. 7.
Effect of ALA-D, RF-hsp 70, or both on the
rate of renaturation of heat-denatured luciferase by hsp 70. Renaturation reactions contained 60 µg/ml ALA-D ( , +), 1.0 µM RF-hsp 70 ( , +), or neither protein ( , ). All
samples but ( ) also received 1.0 µM hsp 70. The final
volume was 110 µl, and 25-µl aliquots were removed at the indicated
times for the determination of luciferase activity. Heat-denatured
luciferase that was not reincubated had 6.5% of the initial activity
of luciferase samples that had not been heated. The results are at
average of two separate experiments.
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Table II
Characterization of the effect of ALA-D on the renaturation of
luciferase by hsp 70
Duplicate samples of heat-denatured luciferase were incubated for 60 min under conditions of renaturation (final volume, 25 µl) and then
assayed for luciferase activity as indicated under "Experimental
Procedures." Unless otherwise indicated, final concentrations were
1.0 µM hsp 70, 60 µg/ml ALA-D, 0.65 µM
RF-hsp 70, 60 µg/ml bovine serum albumin, 60 µg/ml glutathione
S-transferase, 1.0 µM hsp 90, and 3.0 µM
ZnCl2. Added hsp 70 was rabbit reticulocyte unless otherwise
indicated. In the column labeled ADP, renaturation incubations
contained 0.5 mM ADP in place of ATP and received no
creatine phosphate. All values are an average of at least two
experiments, and most are an average of three. ND, = values that were
not determined. Luciferase that was heated and not reincubated had an
average of 6.2% of the activity before heating (range, 5.2-7.1%).
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For comparison, the effect of limiting and saturating amounts of RF-hsp
70 on the renaturation of luciferase in the presence of hsp 70 and
absence of ALA-D is graphed on the ordinate of Fig. 6. RF-hsp 70 produced a just saturating stimulation of renaturation of about 60% of
the initial luciferase activity at 1.0 µM, the same
concentration as the added hsp 70, as we reported previously (1). When
added at 0.25 µM, RF-hsp 70 promoted renaturation to just
under 30% of the original luciferase activity, similar to the effect
of saturating ALA-D. When ALA-D and RF-hsp 70 were added together to
renaturation reactions, they produced exactly an additive effect on
luciferase renaturation, whether each was added at a limiting or
saturating concentration (Fig. 6). Thus, for example, just saturating
concentrations of RF-hsp 70 and ALA-D promoted luciferase renaturation
to 56 and 31% of its initial activity, respectively, when added
separately, and they promoted renaturation to 84% of the original
luciferase activity when added together. This indicates that the effect
of each on luciferase renaturation by hsp 70 is independent of the
other and is probably mediated by separate mechanisms.
We also examined the effect of just saturating levels of ALA-D and
RF-hsp 70 on luciferase renaturation by hsp 70 as a function of time.
The results (Fig. 7) indicate that renaturation of luciferase in the
presence of ALA-D, RF-hsp 70, or both is incubation
time-dependent. Neither (nor both together) increased the
activity of heat-denatured luciferase when added without incubation,
indicating that their effect is at the level of protein renaturation or
folding. The effects of ALA-D and RF-hsp 70 were about additive at
every time point, producing renaturation of luciferase of 26% (ALA-D),
70% (RF-hsp 70), and 85% (both) of the initial activity by 110 min (Fig. 7). Whereas renaturation with ALA-D reached a maximum at 70 min,
renaturation in samples with RF-hsp 70 was still increasing at 110 min.
Results in Figs. 6 and 7 demonstrate that saturating ALA-D and RF-hsp
70 together can produce almost complete renaturation of luciferase by
hsp 70. In contrast, incubation of heat-denatured luciferase by itself
or with only hsp 70 added produces very little renaturation of activity.
We found previously that increasing the hsp 70 concentration above 1.0 µM produces little further increase in luciferase
renaturation by saturating RF-hsp 70 but that below 1.0 µM, hsp 70 becomes limiting (1). Therefore, we compared
the dependence upon hsp 70 concentration of luciferase renaturation in
the presence of ALA-D with that in the presence of RF-hsp 70 (Fig.
8). Renaturation in the presence of
limiting (0.3 µM) RF-hsp 70 requires at least 1.0 µM hsp 70 for a near maximal effect and is only 62 and
23% as great, respectively, with 0.50 and 0.25 µM hsp 70 (Fig. 8B). In contrast, renaturation in the presence of
limiting ALA-D is almost as great with 0.25 µM hsp 70 as
it is with higher hsp 70 concentrations, and even when ALA-D is
saturating, renaturation is still 63% as great with 0.25 µM hsp 70 as it is with 1.0 µM hsp 70 (Fig.
8, A and B). Thus, limiting RF-hsp 70 (0.3 µM) is somewhat more effective at stimulating luciferase
renaturation than saturating ALA-D at 1.0 µM hsp 70 or
above, but it is only one-half as effective at 0.25 µM
hsp 70 (Fig. 8B).

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Fig. 8.
Effect of the concentration of hsp 70 on the
renaturation of heat-denatured luciferase in the presence of ALA-D or
RF-hsp 70. Renaturation reactions (25 µl) contained the
indicated concentrations of ALA-D and 0.25 ( ), 0.50 ( ), 1.0 ( ), or 2.0 ( ) µM hsp 70 (panel A) or the
indicated concentrations of hsp 70 and 33 ( ) or 56 ( ) µg/ml
ALA-D or 0.3 µM RF-hsp 70 ( ) (panel B).
Luciferase activity was measured after a 60-min incubation. Samples of
heat-denatured luciferase which were not reincubated had 5.8% of the
initial activity of nonheated controls, whereas others that were
reincubated but without protein additions had 9.0% of the initial
luciferase activity. The results are an average of three
experiments.
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Additional characterization of the effect of ALA-D on the renaturation
of luciferase by hsp 70 is shown in Table II. When we tested the action
of ALA-D and RF-hsp 70 on renaturation with ADP added in place of ATP
and an ATP-regenerating system, we found that neither produced any
stimulatory effect, in contrast to their activity in the presence of
ATP, confirming that the action of each requires ATP. We also tested
and found that hsp 90, which has a small stimulatory effect on
renaturation by hsp 70, does not enhance renaturation in the presence
of limiting ALA-D or RF-hsp 70 or just saturating ALA-D (Table II).
Other studies have shown that hsp 90 maintains denatured protein in a
folding-competent state (7, 9, 10). In contrast to the dependence upon
Zn2+ of ALA-D enzymatic activity (Fig. 4), the renaturation
effect of just saturating ALA-D is not significantly enhanced by
Zn2+ (Table II). This finding is consistent with the
possibility that these two activities of ALA-D are mediated by
different parts of the protein. Finally, we tested and found that the
degree of renaturation effect produced by ALA-D is dependent upon the
specific hsp 70 added (Table II) because it is most effective with its own (rabbit reticulocyte) hsp 70 (hsc 73 or constitutive form) but only
about one-half as effective with bovine brain hsp 70 (constitutive
form) and less than one-fifth as effective with human hsp 70 (heat-inducible form). We reported previously a similar pattern of hsp
70 specificity with RF-hsp 70 (1).
To determine whether the mechanism by which ALA-D enhances renaturation
by hsp 70 is different from that of RF-hsp 70, we tested the ability of
each or both to promote the dissociation of hsp 70·ADP in the
presence of ATP. We showed previously that RF-hsp 70 binds to hsp 70, lowers the Kd of hsp 70 for ATP to a value that is
close to its Kd for ADP, and thus stimulates the
conversion of hsp 70·ADP to hsp 70·ATP leading to enhanced hsp 70 recycling (1). The results in Fig. 9
demonstrate that the dissociation of hsp 70·[32P]ADP in
the presence of ATP is a relatively slow reaction, and it is not
affected by the addition of limiting (30 µg/ml) or just saturating
(60 µg/ml) concentrations of ALA-D. In contrast, this dissociation
occurs about 4- and 5-fold faster in the presence of limiting (0.5 µM) and just saturating (1.0 µM) RF-hsp 70, respectively, and the stimulation by RF-hsp 70 is not affected by
adding limiting or just saturating ALA-D (Fig. 9). We also tested and
found that just saturating ALA-D has a relatively small effect on the
ATPase activity of hsp 70 (it increases this activity 1.5-fold; data not shown) in contrast to the effect of just saturating RF-hsp 70, which stimulates the ATPase activity of hsp 70 up to 5-fold (1). These
results confirm that RF-hsp 70 and ALA-D promote renaturation by hsp 70 by different mechanisms.

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Fig. 9.
Effect of RF-hsp 70, ALA-D, or both on the
rate of dissociation of hsp 70·[32P]ADP
in the presence of ATP. Samples (25 µl) were constituted
exactly the same as renaturation reactions, but instead of adding
heat-denatured luciferase, the reaction was initiated by the addition
of hsp 70·[32P]ADP, prepared as described previously
(1) (final concentration, 1.0 µM hsp 70). Samples
contained 0.0 (circles), 0.5 (triangles), or 1.0 (squares) µM RF-hsp 70 and 0 (closed
symbols), 30 (half-filled symbols), or 60 (open
symbols) µg/ml ALA-D. Incubation was at 32 °C, and, at the
indicated times, 4.5-µl aliquots were removed and subjected to
Millipore filtration, as indicated (1), to determine the percentage of
the initial hsp 70·[32P]ADP that remained when compared
with zero time samples that were not incubated. The results are an
average of two experiments.
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The Renaturation Stimulatory Activity of ALA-D May Be Caused by a
Region Near Its Carboxyl Terminus Which Has Considerable Sequence
Similarity to the Highly Conserved DnaJ Protein Family
Domain--
Although ALA-D is not a member of the DnaJ protein or hsp
40 family, we examined its primary structure to determine whether any
part of it may show sequence homology to the DnaJ protein family. A
precedent for this is the identification of the brain vesicle-associated protein auxilin as the 100-kDa cofactor that is
required for hsp 70 (hsc 73)-dependent dissociation of
clathrin baskets or clathrin release from coated vesicles (12, 13). The
action of auxilin was found to be dependent upon a carboxyl-terminal domain of the protein (12) which contains considerable sequence similarity to the DnaJ domain, a highly conserved 70-amino acid region
shared by all DnaJ-like proteins that otherwise show considerable sequence variation (12, 13). An examination of ALA-D demonstrates that
it also contains a region near its carboxyl terminus which has
considerable sequence similarity to the DnaJ protein family domain
(Fig. 10). Two different alignments
(upper alone and lower plus right portion of upper) of human ALA-D
(residues 259-296 and 232-296, respectively) with human Hdj-1
(residues 22-53 and 10-53, respectively) demonstrate 29% identity
and 42% similarity between a region near the carboxyl terminus of
ALA-D and the DnaJ domain of Hdj-1. These values are very close to the
similarity between the carboxyl-terminal portion of auxilin and the
DnaJ domain of HSJ1 (22% identity and 52% similarity) or a DnaJ
consensus sequence (32% identity and 50% similarity) (13). They also
are of the same magnitude as the similarity between bacterial DnaJ and
the DnaJ consensus sequence (13). In addition, ALA-D contains the
His-Pro-Asp sequence (at 268-270) that is present in all DnaJ proteins
and has been found to be necessary for the function of DnaJ (29), yeast
Sec63p (30), and yeast YDJ-1 (31). These findings suggest that the
region near the carboxyl terminus of ALA-D may be a DnaJ domain and
that the ability of ALA-D to promote the folding activity of hsp 70, as
shown above, may be caused by an ability to function as a DnaJ
protein.

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Fig. 10.
ALA-D has a presumptive DnaJ domain near its
carboxyl terminus. Two different alignments of a region near the
carboxyl terminus of human ALA-D (46) and the DnaJ domain of human
Hdj-1 (49) are shown. The longer consists of the lower sequence pair
that continues with the right portion of the
upper sequence pair following the vertical
brackets. The single-letter amino acid code is used, identical
residues are marked by a single solid line, and
semiconservative changes are indicated by two dots. Spacing
indicated by the dashes has been used to optimize
alignments.
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DISCUSSION |
This study was prompted in part by the difference between our
characterization of RF-hsp 70, purified from rabbit reticulocyte lysate
(1), and the characterization of the homologous human protein, IEF SSP
3521 (3), expressed as a recombinant protein in, and purified from,
Escherichia coli and termed p60 or hop (6-8). We found that
RF-hsp 70 stimulates the dissociation of ADP from, and the binding of
ATP to, hsp 70, leading to a dramatic increase in the rate of
renaturation of luciferase by hsp 70 (1 and this report). In contrast,
Johnson et al. (8) found that recombinant hop lacks these
very same activities, although it does bind to hsp 70 and hsp 90, and
it does enhance renaturation of luciferase somewhat if YDJ-1 or YDJ-1
and hsp 90 are also added. One possible explanation for this
difference, raised by Johnson et al. (8), is that our RF-hsp
70 may contain contaminants and that hop/RF-hsp 70 itself does not
recycle hsp 70 by promoting adenine nucleotide exchange. In particular,
hsp 40 has been found to contaminate preparations of other proteins
involved in protein renaturation/folding (9, 11), and members of the
DnaJ/hsp 40 protein family do stimulate protein renaturation by hsp 70 (7, 9, 10). Therefore, we tested and found that our RF-hsp 70 is
completely free of hsp 40 (by immunoblot analysis in Fig. 3) and of
trace amounts of any protein migrating faster than the 66-kDa band of
RF-hsp 70 (by loading excess protein on the gel and silver staining in
Fig. 1A). These results demonstrate convincingly that RF-hsp
70 does have the properties of a hsp 70-recycling protein, as we showed
previously (1) and in this report. An alternative explanation,
suggested by Johnson et al. (8) and by us (1), is that, in
contrast to the native cellular protein, recombinant hop/RF-hsp 70 may
be defective, perhaps because this protein, when synthesized in
E. coli, is not folded properly and/or does not undergo
proper disulfide bond formation that may be required for its function.
We believe this alternative remains a distinct possibility. Johnson
et al. (8) also commented that the fact that hop/RF-hsp 70 binds to hsp 70·ADP and not to hsp 70·ATP (1, 8) is inconsistent
with this protein acting as a nucleotide exchange factor, as we have
proposed (1 and this article). We believe, however, that hop/RF-hsp 70 binding to hsp 70·ADP is precisely what would be expected for a
recycling factor that acts by promoting adenine nucleotide exchange. As
discussed previously (1), RF-hsp 70 binding to hsp 70·ADP, either
free or associated with a protein substrate, increases the affinity
(lowers the Kd) of hsp 70 for ATP, leading to a more
rapid rate of dissociation of ADP from, and binding of ATP to, hsp 70 (1). This would increase the rate of recycling of hsp 70, leading to an
increased rate of protein (luciferase) renaturation as observed (1 and
this report) and result, secondarily, in an increased rate of
conversion of ATP to ADP (ATPase activity).
We have also found that rabbit reticulocyte ALA-D, which is about as
active in converting ALA to PBG as is ALA-D from other sources, has the
additional ability to promote protein renaturation by hsp 70. We have
proposed that this additional function may be the result of a region
near the carboxyl terminus of ALA-D which has sequence homology to the
conserved domain of the DnaJ/hsp 40 protein family, suggesting that
ALA-D may act as a DnaJ-like protein, similar to what has been proposed
for the brain protein auxilin (12, 13). Our characterization of the
renaturation effect of ALA-D is most consistent with this hypothesis,
although an alternative explanation for this unexpected action of ALA-D is certainly possible. Like the human DnaJ homolog Hdj-1, which enhances protein renaturation in the presence of hsp 70 but has no
renaturating effect by itself (7, 10), ALA-D stimulates protein
renaturation by hsp 70 and is ineffective by itself. Other DnaJ
homologs, such as YDJ-1 (32) and E. coli DnaJ (for review, see Ref. 33), may have the additional ability to function as molecular
chaperones themselves in the absence of the corresponding hsp 70. It is
thought that this additional function requires the glycine/phenylalanine-rich and the cysteine-rich regions that are
characteristic of only some DnaJ homologs (34). Unlike YDJ-1 and DnaJ,
Hdj-1 and ALA-D lack these regions and possess only the conserved J
domain. We have also found that the degree of renaturation produced by
ALA-D is dependent upon the specific hsp 70 added (Table II). Previous
studies with yeast proteins have also shown specificity in the
cooperative action of individual hsp 70s and DnaJ homologs (32, 35, 36)
and that this is determined by the structure of the individual J domain
(36).
The folding or chaperone function of hsp 70 is regulated by specific
DnaJ homologs, and this may be mediated by the direct interaction of
the J domain of the DnaJ protein with a carboxyl-terminal portion of
hsp 70 (34) which is different from the polypeptide binding domain of
hsp 70 (33). Although the mechanism of how hsp 70 and DnaJ proteins
function together to promote protein folding is not completely clear,
studies using chaperone proteins from E. coli to renature
thermally denatured luciferase in vitro by Schröder
et al. (37) demonstrated that DnaK (hsp 70), DnaJ, and GrpE
were sufficient to produce almost complete renaturation. They found
that DnaJ associated with denatured luciferase and, together with DnaK,
prevented luciferase aggregation, thus permitting subsequent
reactivation. They also demonstrated that the same three proteins were
necessary for renaturation in vivo. Ziemienowicz et
al. (38) demonstrated that these same three proteins can reactivate heat-denatured RNA polymerase, that DnaJ reduced the level
of DnaK required, and that both DnaJ and GrpE are involved in the
formation and the dissociation of a substrate-chaperone complex.
We find it significant that these three E. coli proteins
produce near complete renaturation in vitro, and we obtain
almost complete renaturation with three rabbit reticulocyte proteins, one of which (hsp 70) is homologous to E. coli DnaK and
another of which (ALA-D) may be homologous to E. coli DnaJ.
The third, RF-hsp 70, although structurally related to yeast STI1 (2, 39), has appreciable functional similarity to E. coli GrpE. RF-hsp 70 promotes the release of ADP from hsp 70 in the presence of
ATP (1, Fig. 9), and GrpE stimulates the release of ADP and ATP from
DnaK (40). Although the combination of RF-hsp 70 and ALA-D promotes
almost complete renaturation of heat-inactivated luciferase in the
presence of hsp 70, the action of each with hsp 70 is not dependent
upon the other (Figs. 6 and 7), and each functions by a different
mechanism (Fig. 9). RF-hsp 70 promotes the recycling of hsp 70 by
promoting adenine nucleotide exchange (1), whereas ALA-D, which may act
as a DnaJ homolog, may promote the association as well as the
dissociation of a hsp 70-substrate complex, as envisioned for other
DnaJ proteins. One should also note that rabbit reticulocyte lysate may
contain additional proteins that promote protein folding and
renaturation, such as the large, hetero-oligomeric ring complex TCP-1
(hsp 60 homolog) (41). Similarly, E. coli proteins GroEL
(hsp 60 equivalent) and GroES can reactivate heat-denatured RNA
polymerase by a mechanism that is separate from that of DnaK, DnaJ, and
GrpE (38).
Schumacher et al. (9) found that 0.04-0.10 µM
YDJ-1 produces a maximal stimulatory effect on luciferase renaturation
in the presence of hsp 70, which is much lower than the concentration of ALA-D which is required for maximal effect (60 µg/ml or 1.6 µM relative to the 38-kDa monomer). Schumacher et
al. (9) also reported that Hdj-1 was only 30% as effective as
YDJ-1 in promoting renaturation. One explanation for these differences,
as noted above and in Ref. 9, is that ALA-D and Hdj-1 lack the glycine- and phenylalanine-rich and cysteine-rich domains that are present within YDJ-1 and other but not all DnaJ proteins (34). Thus, Freeman
and Morimoto (10) used 1.6 µM hsp 70 and 3.2 µM Hdj-1 for their renaturation reactions, concentrations
that are similar to the concentrations (1.0 µM hsp 70 and
1.6 µM ALA-D) we have found to be optimal. One additional
consideration is that, unlike other DnaJ proteins, such as E. coli DnaJ, which are thought to function as dimers (33), ALA-D is
an octamer (23, 26, 27), which may require that it be added at a 4-fold
greater molar concentration of the monomer to produce the same effect
of the multimer.
Studies of the enzymes involved in heme biosynthesis have indicated
that in most mammalian tissues, ALA-D is the enzyme whose concentration
is in the greatest excess relative to the others in this pathway (42).
Despite this, the level of ALA-D has been found to increase during
erythroid cell differentiation to a greater degree than several other
heme biosynthetic enzymes whose activities are much less than that of
ALA-D (43-45). Our findings in this report suggest that ALA-D may be
produced in such a great excess in these cells because it can then also
function as a DnaJ protein homolog and promote the protein folding or
chaperone function of hsp 70. We estimate that the concentration of
ALA-D in rabbit reticulocyte lysate is approximately 0.1 mg/ml, which
is close to the concentration of ALA-D (0.06 mg/ml) we have found to be just saturating in promoting the renaturation of luciferase by hsp 70 in an isolated reaction. Although rabbit reticulocytes do contain
DnaJ/hsp 40 protein (9, Fig. 3), our findings suggest that this protein
is much less abundant than ALA-D (data not shown). This suggests that
ALA-D may be the major functional DnaJ protein in rabbit reticulocytes
and that this may be why it is produced in such great excess during
erythroid cell differentiation.
 |
ACKNOWLEDGEMENT |
We are grateful to Giri Reddy for performing
the amino acid sequencing determinations.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant HL-30121.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.
To whom correspondence should be addressed: Dept. of Pathology,
The University of Chicago, 5841 South Maryland, Chicago, IL 60637. Tel.: 773-702-0827; E-mail: mgross{at}midway.uchicago.edu.
The abbreviations used are:
hsp 70, a
member of the heat shock protein 70-kDa family; hsc 73, the
constitutive form of hsp 70; hsp 72, the heat-inducible form of hsp 70; hsp 90, a member of the heat shock protein 90-kDa family; DnaJ/hsp 40, a member of the DnaJ heat shock protein 40-kDa family; RF-hsp 70, a
66-kDa rabbit reticulocyte protein that recycles hsp 70 (this protein
has also been termed p60 and hop); ALA,
-aminolevulinic acid; ALA-D,
-aminolevulinic acid dehydratase; PBG, porphobilinogen; ProHCR, the
latent precursor of the heme-regulated, eIF-2
kinase (HCR or HRI); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
 |
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