(Received for publication, May 17, 1995; and in revised form, July 17, 1995)
From the
The precursor (pmAspAT) and mature (mAspAT) forms of
mitochondrial aspartate aminotransferase interact with hsp70 very early
during translation when synthesized in either rabbit reticulocyte
lysate or wheat germ extract (Lain, B., Iriarte, A., and
Martinez-Carrion.(1994) J. Biol. Chem. 269,
15588-15596). The nature of the structural elements responsible
for recognition and binding of this protein to hsp70 has been studied
by examining the folding and potential association with the chaperone
of several engineered forms of this enzyme. Whereas pmAspAT and mAspAT
bind hsp70 very early during translation, the cytosolic form of this
enzyme (cAspAT) does not interact with hsp70. A fusion protein
consisting of the mitochondrial presequence peptide attached to the
amino terminus of cAspAT associates with hsp70 only after the protein
has acquired its native-like conformation, apparently through binding
to the presequence exposed on the surface of the folded protein.
Deletion of the amino-terminal segment of mAspAT or its replacement
with the corresponding domain from the cytosolic isozyme eliminates the
cotranslational binding of hsp70 to the mitochondrial protein. We
conclude that both the presequence and NH-terminal region
of pmAspAT represent recognition signals for binding of hsp70 to the
newly synthesized mitochondrial precursor. Results from competition
studies with synthetic peptides support this conclusion. The ability of
hsp70 to discriminate between these two highly homologous proteins
probably involves the recognition of specific sequence elements in the
NH
-terminal portion of the mitochondrial protein and may
relate to their separate localization in the cell. A slower folding
rate and higher affinity for cytosolic chaperones may represent
evolutionary adaptations of translocated mitochondrial proteins to
ensure their efficient importation into the organelle.
Many denatured proteins are able to refold spontaneously which
clearly demonstrates that the functional three-dimensional structure of
proteins is solely determined by their amino acid sequences (Anfinsen
and Scheraga, 1975; Jaenicke, 1991). In order to avoid the competing
reactions of aggregation, low temperatures and low protein
concentrations are used in most of these in vitro refolding
studies. However, in vivo, proteins manage to fold at high
protein concentrations and at physiological temperatures (37 °C).
Apparently, this is accomplished with the collaboration of external
factors such as molecular chaperones, which control the process by
binding to incompletely folded polypeptides and thereby preventing
their aggregation, misfolding or premature folding (for recent reviews,
see Gething and Sambrook(1992) and Hendrick and Hartl(1993)).
Furthermore, in the intact cell, proteins may initiate folding during
biosynthesis on the ribosome and before the polypeptide chain is
released into the cytoplasm (Freedman, 1992; Fedorov et al.,
1992; Kolb et al., 1994; Fedorov and Baldwin, 1995). This
cotranslational folding is probably assisted by molecular chaperones.
Chaperones DnaJ (Hendrick et al., 1993) and hsp70 ()(Hansen et al., 1994) have been found associated
with newly synthesized proteins still attached to the ribosome. Frydman et al.(1994) recently proposed that firefly luciferase
synthesized in rabbit reticulocyte lysate (RRL) interacts sequentially
with hsp40 (a homologue of bacterial DnaJ), hsp70, and TCP-1 containing
ring complex. These interactions allow the cotranslational formation of
protein domains and contribute to the rapid folding of the newly
synthesized enzyme once released from the ribosome (Fedorov and
Baldwin, 1995).
The extent of folding achieved before the polypeptide chain is released from the ribosome may, however, vary for different proteins. For instance, the precursors of translocated mitochondrial proteins must be maintained in a partially unfolded, loose conformation in the cytosol in order to be imported into the organelle (Verner and Schatz, 1987; Pfanner et al., 1987). Such a delayed folding may not be required for proteins that stay in the cytosol, and therefore they might afford to fold rapidly after synthesis. The mechanism by which cells prevent the ``premature'' folding of proteins before being translocated across the mitochondrial membranes is not fully understood, but various proteins, including cytosolic hsp70, have been implicated in the process (Chirico et al., 1988; Deshaies et al., 1988; Murakami et al., 1988; Sheffield et al., 1990).
The cytosolic and mitochondrial isozymes of aspartate
aminotransferase (AspAT) constitute a well suited system to investigate
the factors responsible for the selective control of the folding of
proteins according to their final destination in the cell. Both enzymes
are encoded by the nuclear DNA and synthesized in cytosolic polysomes.
The two dimeric proteins share a high degree of sequence homology, an
almost superimposable three-dimensional structure, and a basically
identical catalytic mechanism (Jansonius and Vincent, 1987). However,
they show differences in overall stability (Iriarte et al., 1984c) and isoelectric point (pI). The more stable cytosolic
enzyme (cAspAT) has a slightly acidic pI, whereas the mitochondrial
form (mAspAT) has a pI of around 9.0 (Martinez-Carrion and Tiemeier,
1967). Furthermore, the folding process of the two isozymes synthesized
in cell-free extracts is clearly distinctive. The mitochondrial enzyme,
which is synthesized in the cytosol as a precursor protein with a
29-residue amino-terminal extension or presequence peptide (pmAspAT;
Altieri et al., 1989; Mattingly et al., 1987), folds
relatively slowly after in vitro synthesis in RRL (half-life
of 100 min). By contrast, the highly homologous cytosolic isozyme
rapidly acquires a protease-resistant conformation after synthesis in
the same cell-free extract (Mattingly et al., 1993a, 1993b).
Yet, the spontaneous in vitro refolding of the cytosolic
enzyme after denaturation with guanidine hydrochloride is only
2-3-fold faster than the mitochondrial enzyme (Iriarte et
al., 1994; Mattingly et al., 1995). It is thus apparent
that components of the cell-free extracts affect differently the
folding rate of each isozyme.
We recently reported that hsp70 interacts with both the precursor and mature forms of mitochondrial AspAT very early during translation in either RRL or wheat germ extract (Lain et al., 1994). In this work we have continued these studies using the cytosolic isozyme of AspAT and several chimeric constructs prepared by exchanging selected sequence fragments between the two isozymes. Both the rate of folding and the interaction with chaperones were analyzed for these diverse AspAT forms synthesized in cytosolic-like environments. We found that the distinct folding rate of the cytosolic and mitochondrial members of this isozyme pair may be determined by the exclusive interaction of chaperones with the mitochondrial form. In addition, this study provides information regarding the targeting signals that allow molecular chaperones to discriminate between highly homologous proteins destined to reside in different intracellular compartments.
Antibodies against hsp70 were raised in rabbits by hyperimmunization with purified bovine brain hsp70 (Welch and Feramisco, 1985) emulsified with Freund's complete (primary injection) or incomplete adjuvant. IgG were purified using a protein A-agarose (Repligen) column or a DEAE-Affi-Gel blue column (Bio-Rad). Antibodies were used either as whole serum or as purified IgG.
pBSKS-4 was used as a
template to generate by PCR a cDNA sequence spanning the presequence
and the first 30 residues of the NH-terminal region of
pmAspAT. We used the ULTma(TM) DNA polymerase (Perkin-Elmer), since
it has been described that this enzyme does not incorporate any
noncoding bases. (
)As a reverse primer
CATCTTCTTGCTGTTGGTATCTCTC that matches from position 5`-251 to position
3`-276 of pBSKS-4 (GeneBank(TM) accession number M18467, Mattingly et al., 1987) was used. As forward primer we used the
oligonucleotide CGGAATTAACCCTCACTAAAG which corresponds to the T3
promoter region of pBluescript KS. The PCR product contained the
sequence of pBluescript, including KpnI to EcoRI
restriction sites from pBluescript KS multiple cloning site, and the
nucleotides encoding amino acid residues 1 through 29 of the
presequence peptide and the first 30 amino acid residues of the
NH
-terminal region of mAspAT. This PCR product was digested
with SalI, and the 300-bp DNA fragment was purified by
electroelution. The sticky blunt-end fragment was ligated to the
4787-bp fragment described above. The desired recombinant plasmid,
pBSKS-9, was selected by PCR screening from randomly selected
transformants using the primers described above and was subsequently
sequenced.
A complete cDNA encoding mcAspAT was assembled from
plasmids pBSKS-5, encoding mAspAT, and pBSKS-6, encoding pcAspAT.
pBSKS-5 was used as a template for the PCR reaction using the same
primers described above. The PCR product included the nucleotides
encoding the first 30 amino acids (residues 3-33) from the
NH-terminal region of the mitochondrial mAspAT protein.
After digestion with SalI, the 221-bp DNA fragment was
purified by electroelution and ligated to the 4787-bp HpaI-SalI fragment from pBSKS-6. The desired
recombinant plasmid, pBSKS-10, was selected by PCR screening from
randomly selected transformants and was subsequently sequenced.
The pcmAspAT chimera was constructed by combining segments of the pBSKS-6 and pBSKS-4 cDNAs using recombinant PCR. The primer PCR1 (GATGATCCCGATCCCCGCAAGGTTAACCTGGGAGTTGGTG-CCTA) defines the splice point between the two proteins. It encodes the last 8 amino acids of the 63-residue amino-terminal pcAspAT peptide to be attached to the mAspAT core protein; the underlined nucleotides encode pmAspAT, beginning at nucleotide 276 which begins the codon for amino acid 63. Primer PCR2 (ATGTCAAAGAATGCGAAGAGAT) is complementary to nucleotides 826-805 in the coding strand for pmAspAT. As PCR3 we used the primer corresponding to the T3 promoter region of pBluescript KS indicated before; PCR4 (AACCTTGCGGGGATCG) is complementary to nucleotides 314-291 which encode the last 8 amino acids of the peptide to be attached to the mAspAT core. The two fragments were freed of excess primers by ultrafiltration, aliquots of each were combined, and another amplification using PCR3 and PCR2 was performed. The resulting product was digested with NdeI and PstI and the desired 340 bp was gel-purified. This fragment was ligated to NdeI- and PstI-digested pBSKS-4 to yield pBSKS-8. Further information on these constructs is available from the authors upon request.
Figure 1:
Diagram
illustrating the amino-terminal regions of the proteins used in this
study. These amino acid sequences are deduced from the DNA sequences of
cDNAs isolated for the cytosolic and the precursor to mitochondrial
aspartate aminotransferase from rat liver (GeneBank(TM) accession
numbers M18467 for pmAspAT and J05263 for cAspAT). The shaded box represents the cytosolic protein, the empty box represents the mitochondrial mature protein, and the hatched
box represents the mitochondrial presequence peptide (residues
numbered -1 to -29 starting at the junction with the mature
moiety). The amino-terminal residue of mAspAT is designated as residue
number 3 to maximize the alignment with the cytosolic isozyme sequence
which contains two additional residues at the NH-terminal
end.
The various protein forms were synthesized in RRL at 30
°C, as previously reported (Lain et al., 1994), using
[S]methionine as the radiolabeled amino acid.
All of them migrated in SDS-PAGE gels according to their expected
molecular weights (data not shown). The constructs which contain the
core component from the cytosolic isozyme, cAspAT, pcAspAT, pmcAspAT,
and mcAspAT, are more stable in RRL after synthesis at 30 °C than
their mitochondrial counterparts (Mattingly et al., 1993b;
data not shown). Furthermore, the yield of protein synthesis for
pmcAspAT and mcAspAT is about 3-fold higher than for the mitochondrial
and cytosolic wild type proteins. The reasons for this difference in
translation efficiencies are unknown.
The association of the different constructs with hsp70 during protein synthesis in RRL (Fig. 2) or upon further incubation following termination of the translation reaction (Fig. 3), was followed by analyzing at different times the total amount of protein present and the amount of radioactive translation product coimmunoprecipitating with anti-hsp70 antibodies. As shown before (Lain et al., 1994), both the precursor and mature forms of mitochondrial AspAT coimmunoprecipitate with hsp70 at the early stages of the translation reaction. The fraction of pmAspAT and mAspAT associated with hsp70 decreases rapidly as translation progresses (data included in Fig. 2A for comparison purposes), suggesting a ``transient'' association of the translation product with the chaperone. The amount of pmAspAT coimmunoprecipitating with hsp70 increases again when the translation reaction is incubated at 20 °C following completion of protein synthesis (Fig. 3B). This ``stable'' reassociation of hsp70 with pmAspAT is dependent on the presence of the presequence and parallels the acquisition of protease resistance by the translation product (Fig. 3A). Apparently, hsp70 binds tightly to the presequence exposed on the surface of the folded protein. By contrast, no coimmunoprecipitation of cAspAT with hsp70 antibodies could be detected at the earliest times of the protein synthesis reaction that we were able to analyze (Fig. 2B). This result indicates that there may be selectivity in the interaction of even highly homologous proteins with chaperones.
Figure 2:
Association of hsp70 with in vitro translated mAspAT and cAspAT constructs during protein synthesis.
Immunoprecipitation of radiolabeled translation products after
initiation of protein synthesis was done by removing, at different
times after initiation of translation, one 2.5-µl aliquot and one
5.0-µl aliquot from RRL translation reactions programmed with mRNA
either for mAspAT (A) or cAspAT (B) variants. After
addition of cycloheximide to stop protein synthesis, the 2.5-µl
aliquot was used to estimate the amount of protein synthesized at each
time, and the 5-µl aliquot was subjected to immunoprecipitation
with polyclonal anti-hsp70 antibodies (Lain et al., 1994). The
amount of radiolabeled translation product synthesized or
immunoprecipitated was determined from the intensity of the
corresponding radioactive band following SDS-PAGE analysis of the
samples and exposure to a PhosphorImager(TM) screen. The fraction of
translation product immunoprecipitated is expressed as percentage
relative to the total amount of protein that is synthesized and is the
average of two determinations. Symbols are as follows: A,
, pmAspAT;
, mAspAT;
,
3-30 mAspAT; and
, pcmAspAT; B,
, pmcAspAT;
, mcAspAT or
cAspAT;
, pcAspAT.
Figure 3: Trypsin resistance and association with hsp70 of different mAspAT and cAspAT protein forms synthesized in RRL after further incubation at 20 °C. Proteins were synthesized in RRL as described under ``Experimental Procedures'' (20 min, 30 °C). After stopping protein synthesis by addition of cycloheximide and chilling on ice, the translation reactions were transferred to a 20 °C water bath. Aliquots were removed either immediately (t = 0 min) or after 240-min incubation (t = 240 min) and analyzed for acquisition of trypsin resistance (A) and for coimmunoprecipitation with hsp70 antibodies (B) as described previously (Lain et al., 1994). Please notice that the zero time (t = 0 min) in this figure follows the last time point in Fig. 2immediately after arresting protein synthesis. Data are expressed as the percentage of total labeled pmAspAT present (aliquot not treated with trypsin) that has become trypsin-resistant (aliquot treated with trypsin) (A) or that immunoprecipitates with hsp70 antibodies (B).
To further asses the role of the presequence peptide in the interaction with hsp70, we studied whether the fusion protein pcAspAT, which has the mitochondrial presequence fused to the amino-terminal region of the cytosolic protein, associates with hsp70. At the early stages (4 min) of the translation reaction, no protein was found to immunoprecipitate with hsp70 antibodies. However, as the translation reaction proceeds, the amount of pcAspAT coimmunoprecipitating with hsp70 progressively increases (Fig. 2B) and remains steady during further incubation of the translation product after stopping protein synthesis (Fig. 3B). Similar results were obtained with pmcAspAT, another cytosolic construct containing the presequence peptide. Since folding of these cytosolic-like proteins is almost completed by the time the translation reaction is interrupted (Fig. 3A), this raise in stable hsp70 binding probably reflects the increase in accessibility of the presequence peptide as the translation product acquires its native conformation, as mentioned before for the natural mitochondrial precursor.
Thus, it is clear that the presequence of
pmAspAT represents a ``linear epitope'' for recognition by
and binding to hsp70, at least in the folded precursor proteins.
However, and quite paradoxically, the presence of this sequence
apparently is not sufficient for binding of this chaperone to the
polypeptide chain while it is emerging from the ribosome. Additional
recognition signals in the mature portion of the mitochondrial protein
might be responsible for this early interaction. One possible candidate
is the NH-terminal region of the mature protein which in
the native enzyme interacts with the surface of the other subunit and
contributes to the stability of the dimer (McPhalen et al.,
1992). This NH
-terminal segment shows a very low similarity
score when compared with the corresponding region of cAspAT (Mattingly et al., 1993b). As shown in Fig. 2A, deletion
of the 28-residue NH
-terminal fragment of mAspAT
(
3-30 mAspAT), or its substitution with the corresponding
sequence from cAspAT (pcmAspAT), dramatically decreased the amount of
translation product immunoprecipitating with hsp70 antibodies during
the initial stages of the translation reaction. Binding of hsp70 to the
presequence-containing pcmAspAT did not increase either upon incubation
at 20 °C after arresting translation (Fig. 3B),
probably because this protein, like
3-30 mAspAT, is unable
to acquire a folded, protease-resistant conformation (Fig. 3A). According to these results, the
NH
-terminal peptide of mAspAT appears to be essential not
only for binding to hsp70 but also for productive folding of the newly
synthesized polypeptide chain.
In order to clarify the involvement
of the NH-terminal region of mAspAT on its interaction with
hsp70, we examined the consequences of introducing this mAspAT region
into the NH
-terminal end of cAspAT. The hypothesis
underlying the design of these experiments was that if that sequence
constitutes a linear recognition site for hsp70, it should be able to
render the otherwise inert cAspAT a ``substrate'' for the
chaperone, as the presequence does for the folded protein. Yet, the
data presented in Fig. 2B show that the level of
immunoprecipitation of mcAspAT early during translation is identical to
that of wild type cAspAT. When the NH
-terminal sequence was
inserted together with the presequence peptide (pmcAspAT), the levels
of coimmunoprecipitation at the initial translation times were still
identical to the corresponding wild type control (pcAspAT in this
case). Only after incubation following the end of translation, this
presequence-containing protein appeared in a complex with hsp70 (Fig. 3B). This association correlated once more with
the appearance of trypsin resistant species (Fig. 3A).
Thus, the presequence and the NH
-terminal peptides of
pmAspAT by themselves do not contain enough information to transform
the cAspAT chains emerging from the ribosome into a target recognizable
by hsp70. Either additional targeting signals are required for binding
or the rate of folding of the protein being synthesized determines the
accessibility of the potential binding sites to interaction with the
chaperone.
Figure 4:
Effect of cAspAT and pmAspAT
NH-terminal peptides on the yield of protein synthesis in
RRL. Approximately 74 µM final concentration of the
different synthetic peptides was added to the RRL translation reactions
before initiation of protein synthesis by the addition of mRNA coding
for pmAspAT (A), cAspAT (B), or brome mosaic virus (BMV) protein. The same amount in volume of DEPC water was
added to the control reactions in the absence of peptides. At different
times, aliquots were taken and, after SDS-PAGE electrophoresis, the
amount of protein synthesized at each time was estimated from the
amount of radiolabel present in the corresponding electrophoretic band.
Symbols are:
, control without peptide;
, mitochondrial
NH
-terminal peptide;
, cytosolic
NH
-terminal peptide; and
, presequence
peptide.
To rule out the possibility that the decrease in protein synthesis caused by this peptide was due to the presence of RNase activity in the peptide solution, we checked the stability and integrity of the pmAspAT mRNA in a mocking translation reaction containing peptide. The results showed that the addition of peptide, even at concentrations higher than those used in the complete translation reaction, did not produce detectable degradation of mRNA (data not shown). Hence, we can conclude that these mitochondrial peptides inhibit protein synthesis by competing for some component(s) of the protein synthesis machinery.
The inhibition of protein
synthesis caused by the mitochondrial synthetic peptides made the
analysis of the binding of hsp70 to the newly synthesized protein under
these conditions difficult, which initially was our main objective. The
almost undetectable levels of pmAspAT synthesized in the presence of
the presequence peptide precluded any further analysis of the
translation product. The mitochondrial NH-terminal peptide
caused a slight decrease in the fraction of protein
coimmunoprecipitating with hsp70 early during translation (data not
shown), but the low quantities of protein synthesized hindered the
accuracy of the measurements. As expected, the cytosolic
NH
-terminal peptide did not have any effect on the level
and time course of the coimmunoprecipitation of pmAspAT during protein
synthesis (data not shown). We also examined whether any of these
synthetic peptides was able to prevent the more stable binding of hsp70
to the folded, trypsin-resistant pmAspAT. The peptides were added after
stopping the synthesis of pmAspAT with cycloheximide. Then the folding
of the translation product and its association with hsp70 were followed
during prolonged incubation of the sample at 20 °C. Both the
mitochondrial presequence and NH
-terminal peptides
diminished significantly the fraction of pmAspAT immunoprecipitated
with hsp70 antibodies (Fig. 5). This inhibition was dependent on
the concentration of peptide present and at
150 µM prevented almost completely the binding to hsp70. This effect was
much less pronounced for the cytosolic NH
-terminal peptide (Fig. 5) which caused a partial decrease in immunoprecipitation
only at the highest concentration used. These findings corroborate that
hsp70 shows a clear preference for binding of the presequence and
NH
-terminal peptides of the mitochondrial isozyme.
Figure 5:
Effect of post-translational addition of
presequence and NH-terminal peptides on the association of
hsp70 with pmAspAT translation product. pmAspAT was synthesized in RRL
for 20 min at 30 °C as described under ``Experimental
Procedures.'' After stopping protein synthesis, different
concentrations of the mitochondrial NH
-terminal peptide
(
, 86 µM;
, 146 µM), the cytosolic
NH
-terminal peptide (
, 86 µM;
,
146 µM), the presequence peptide (
, 92
µM;
, 157 µM), or an equivalent
volume of DEPC-treated water (
, control) were added to the
translation reactions. The reactions were incubated further at 20
°C to allow the translation product to acquire a trypsin-resistant
conformation. At different incubation times, aliquots were taken and
immunoprecipitated with hsp70 antibodies as described in the legend to Fig. 2.
Binding of hsp70 to the presequence is not necessary for proper
folding of the pmAspAT translation product in RRL. Neither the
presequence nor the cytosolic NH-terminal peptide affect
the folding of pmAspAT in RRL (Fig. 6). However, the
mitochondrial NH
-terminal peptide causes a marked decrease
in the yield of protease-resistant pmAspAT. This effect is most likely
due to a direct interference of this peptide with folding of the
polypeptide chain rather than to depletion of the chaperone in the
lysate. As indicated before, this region of the molecule appears to be
critical for proper folding of the mitochondrial enzyme. During in
vitro refolding studies of the acid-unfolded pmAspAT, we could
observe that addition of the NH
-terminal peptide to the
refolding mixture inhibited dramatically the spontaneous reactivation
of the enzyme. (
)The mechanism of this inhibition is not
known.
Figure 6:
Effect of post-translational addition of
presequence and NH-terminal peptides on the folding of
pmAspAT after synthesis in RRL. pmAspAT was synthesized in RRL as
described under ``Experimental Procedures.'' After stopping
protein synthesis, approximately 86 µM of the synthetic
peptides, or an equivalent volume of DEPC water, were added to the
translation mixtures. Samples were incubated at 20 °C, and folding
of the translation product was followed by monitoring acquisition of
trypsin resistance. Data are expressed as the percentage of total
labeled pmAspAT present (aliquot not treated with trypsin) that has
become trypsin-resistant (aliquot treated with trypsin). Symbols are:
, control in the absence of peptides;
, mitochondrial
NH
-terminal peptide;
, cytosolic
NH
-terminal peptide; or
, presequence
peptide.
It is now widely accepted that protein folding and assembly in the cell are mediated by molecular chaperones (Gething and Sambrook, 1992; Hendrick and Hartl, 1993). However, very little is known regarding the mechanism of action of these chaperones in vivo, mainly due to the scarcity of suitable analytical procedures and the complexities of the experimental systems under study. Several questions still remain open: are there any differences in the control of the folding processes of cytosolic and membrane translocated proteins?; what determines whether a particular chaperone interacts with a nascent protein?; and which features of a newly synthesized polypeptide chain target it for recognition by a particular chaperone or set of chaperones?
In this work we address some of these
questions using the mitochondrial and cytosolic isozymes of aspartate
aminotransferase. These two homologous proteins are synthesized on
cytosolic free polysomes. After translation, cAspAT remains in the
cytosol, whereas mAspAT is translocated into the matrix of
mitochondria. As for most translocated mitochondrial proteins (Pfanner et al., 1994; Verner, 1993), efficient uptake of mAspAT by
mitochondria requires the presence of the NH-terminal
presequence peptide as well as an incompletely folded conformation
(Mattingly et al., 1993a). From the latter requirement it
follows that the folding of the newly synthesized protein must be slow
enough to allow it to engage the import machinery in a competent
conformation. Indeed, as we reported earlier, folding of the
mitochondrial precursor synthesized in cell-free extracts proceeds at a
slow rate (t
> 1 h at 15 °C) (Mattingly et al., 1993a). If mitochondria are introduced into the system
immediately after finishing translation, the protein is imported into
the organelle before it has a chance to fold into a protease-resistant
conformation. In contrast, the cytosolic isozyme folds markedly faster
under the same conditions (Mattingly et al., 1993b). How these
two isozymes evolved to display such different folding rates in
cytosolic-like environments is not known, but their distinct behavior
may relate to their separate locations in the cell.
What determines
the different folding rates of these two proteins after synthesis in
cell-free extracts? Since the rates of refolding in buffer of the
chemically denatured purified proteins are very similar (Mattingly et al., 1995), selective interaction with molecular chaperones
is an obvious possibility. The findings presented in this work support
this hypothesis, at least regarding hsp70. This chaperone could be
detected associated with the mitochondrial translation product (Lain et al., 1994), whereas no such complex was found with the
cytosolic form. From these data one can tentatively infer that perhaps
cytosolic proteins do not need the collaboration of molecular
chaperones for the regulation of their intracellular folding, because
translocation through a membrane system is not part of their maturation
process. In fact, mAspAT and cAspAT also show very different behavior
with regard to their interaction with chaperones during in vitro refolding of the chemically denatured proteins. hsp70 arrests
folding of acid-unfolded mAspAT but not cAspAT. Furthermore, the Escherichia coli chaperonin groEL
completely stops the refolding of mAspAT at low temperatures, but only
slows down the reaction for cAspAT (Mattingly et al., 1995).
Interestingly, the cytosolic enzyme is able to refold from its
guanidine HCl unfolded state at physiological temperature in the
absence of chaperones. By contrast, mAspAT, perhaps because of a higher
tendency of some of its folding intermediates to aggregate, requires
the assistance of groEL/groES to refold at 37 °C after chemical
denaturation (Mattingly et al., 1995).
Similar findings have been reported for the cytosolic and mitochondrial isozymes of malate dehydrogenase (Stanford et al., 1994). For this enzyme, a correlation was proposed between the overall hydrophobicity of the isozyme and the strength of its interaction with groEL. Mitochondrial malate dehydrogenase, which binds more tightly to groEL, has a higher hydrophobic potential than the cytosolic isozyme (Stanford et al., 1994). However, no such correlation exists for mAspAT and cAspAT. Both enzymes contain about equal amounts of hydrophobic amino acids (Mattingly et al., 1995) very similarly distributed along their primary structure (Mattingly et al., 1993b). Thus, this feature alone cannot explain the different affinity of these enzymes' folding intermediates for chaperones. Perhaps the slightly greater spontaneous refolding rate of cAspAT is responsible for those differences. The rapid collapse of the unfolded protein to a compact structure could prevent a strong interaction with hsp70 which is known to favor binding of highly unstructured or extended regions of polypeptide chains (Flynn et al., 1991; Hartl et al., 1994; Palleros et al., 1994). Other chaperones, such as groEL, may recognize instead certain conformational features of intermediates in the folding pathway of the protein.
Are there linear sequence
motifs that are particularly important for recognition and binding to
hsp70? Previous results with the precursor and mature forms of mAspAT
suggested that the presequence peptide may be one of the structural
elements that are involved in this interaction (Lain et al.,
1994). In addition, recombinant chicken pmAspAT expressed in E.
coli copurifies with hsp70 (DnaK) tightly attached to its
presequence peptide (Schmid et al., 1992). Our results with
the fusion protein pcAspAT agree with these observations. Since cAspAT
does not bind hsp70, the interaction of the chaperone with
trypsin-resistant pcAspAT must involve the presequence peptide fully
accessible in the folded protein. Several pieces of indirect evidence
indicate that the presequence peptide lies exposed on the surface of
native pmAspAT or pcAspAT precursors. For example, the peptide is
extremely susceptible to trypsin hydrolysis (Martinez-Carrion et
al., 1990). Furthermore, the presence of the presequence does not
affect either the catalytic activity or the folding of the mature
moiety of the proteins. Hence, although we do not know the exact
arrangement of this peptide in the three-dimensional structure of the
precursors, we envision it as an appendix at the
NH-terminal end with only minimum, if any, contact with the
surface of the protein dimer. Consequently, binding of hsp70 to the
presequence peptide does not prevent the folding of the protein either.
However, if the presequence peptide in the folded proteins represents a linear targeting signal for hsp70, why were we unable to detect the transient complex of hsp70 with presequence-containing cAspAT forms during translation? One possibility is that the presequence is not available for recognition by hsp70 when the protein is emerging from the ribosome. More likely, the complex may form, but due to the rapid folding of the elongating cAspAT chain, the chaperone may dissociate during the dead time of our method of analysis. Additional investigation of the folding state of the cytosolic enzyme during translation would be required to clarify this point.
Since
mature mAspAT, lacking the presequence peptide, also
coimmunoprecipitates with hsp70 antibodies early during translation, it
is evident that the mature part of the mitochondrial protein contains
additional recognition signals for the chaperone. We focused our
attention initially on the NH-terminal region of the mature
protein for several reasons. First, since protein synthesis proceeds
from the amino- toward the carboxyl-terminal end of the chain, this
region would be the first to emerge from the ribosome immediately after
the presequence peptide. Second, the low degree of sequence homology on
this region of the two isozymes makes it a likely target for selective
recognition of mAspAT by hsp70. Finally, appropiate restriction sites
exist in the area which facilitates the manipulation of the parent cDNA
in the preparation of chimeras.
Despite these predictions, the
results from our studies on this sequence region are inconclusive. On
the one hand, the deletion of this segment or its substitution with its
equivalent from cAspAT eliminates the initial coimmunoprecipitation of
the translation product with hsp70 antibodies during protein synthesis.
The results obtained from competition studies using synthetic peptides
corroborate that hsp70 shows a clear preference for binding to the
mitochondrial NH-terminal (and presequence) peptides
relative to the corresponding cytosolic sequence. However, its
incorporation into the NH
-terminal region of the cytosolic
enzyme fails to convert this isozyme into a target for hsp70. It is
likely, therefore, that the association of mAspAT with hsp70 involves
other regions of the protein structure besides the
NH
-terminal peptide, probably with the participation of
several chaperone molecules binding to different recognition signals
along the polypeptide chain.
On the other hand, folding of mAspAT is
drastically affected by deletion or replacement of its
NH-terminal arm. Furthermore, only the mitochondrial
NH
-terminal peptide interferes with folding of pmAspAT,
even when added posttranslationally. These results suggest that proper
fitting of the first 10 residues at the NH
-terminal end of
one subunit into the hydrophobic pocket on the surface of its
neighboring monomer is essential for efficient folding and/or assembly
of the mAspAT dimer. The synthetic peptide probably competes for the
same pocket on the surface of the protein that usually accepts the
first 10 residues of the NH
-terminal segment of the chain.
This anchoring contributes to the stabilization of the quaternary
structure of the enzyme (McPhalen, 1992) and is also essential for the
open-closed conformational transitions that accompany catalysis in both
isozymes (Iriarte et al., 1984b). However, similar alterations
in the corresponding region of cAspAT only decreases slightly the yield
of folding in RRL (Fig. 3A), suggesting that the
subunit interaction involving the NH
-terminal arm is not as
critical for dimerization of this isozyme. These observations correlate
with our earlier reports showing that after limited trypsin cleavage,
the NH
-terminal peptide is easily removed from cAspAT
(Iriarte et al., 1984a) but remains tightly associated with
the protein core in mAspAT, (
)suggesting a much stronger
interaction at this subunit contact area in the mitochondrial protein.
The significance of this difference in association energy between the
two isozymes at this region of the subunit interface is not clear.
Perhaps the most surprising observation reported in this work is the
dramatic effect of the mitochondrial peptides, particularly the
presequence, on the synthesis of the mitochondrial and cytosolic AspAT
as well as of the unrelated brome mosaic virus protein. The cytosolic
peptide, however, is completely innocuous. Apparently, the
mitochondrial peptides are competing with the nascent polypeptide
chains for binding to some component of the protein synthesis machinery
of RRL essential for completion of translation. Although the
information available is insufficient to identify this factor(s), we
can speculate that perhaps hsp70, which can bind the presequence and
the mitochondrial NH-terminal peptides, is at least one of
them. The interaction of this chaperone with a variety of nascent
proteins while still attached to the ribosome is well documented
(Beckmann et al., 1990; Hansen et al., 1994). It has
been suggested that hsp70 may even have an active role in translation
by aiding in the movement of the elongating chain through the ribosome
channel, analogous to its role in protein translocation across
membranes (Nelson et al., 1992). Consequently, depletion of
free hsp70 by introduction of an excess of a suitable ligand, such as
the presequence peptide of pmAspAT, would inhibit efficient protein
synthesis.
In any case, this study suggests that hsp70 may not be
directly involved in the slow posttranslational folding observed for
mAspAT in RRL (Mattingly et al., 1993a). It may instead play a
more essential role in the successful completion of the translation
process or in the degree of cotranslational folding that the
polypeptide chain achieves. The affinity of hsp70 for certain binding
sites in the nascent chain, perhaps in combination with the intrinsic
folding rate of the chain, could determine the stability and hence
longevity of the initial complex. The two phenomena might be actually
connected. It is possible that low affinity binding of hsp70 to the
emerging cAspAT chain occurs and is sufficient to aid in the elongation
of the chain, but not to slow down the intrinsically fast
(cotranslational) folding of the translation product. This in turn
would prevent binding of additional cytosolic factors, and the protein
could finish folding quickly without assistance from other proteins. In
contrast, a stronger association of hsp70 with the
NH-terminal region of the nascent pmAspAT might slow down
the initial acquisition of structure, thus allowing the subsequent
binding of additional hsp70 molecules and/or other cytosolic components
to recognition signals or hydrophobic regions that become transiently
exposed in the elongating chain. Folding of the completed translation
product after its release from the ribosome would then require its
dissociation from this initial complex with chaperone(s), perhaps with
the participation of other soluble cytosolic components (Lain et
al., 1994; Mattingly et al., 1993a).
In summary, the interaction of molecular chaperones with nascent proteins is not solely by specific linear sequence elements. To transform proteins from low affinity or nonbinding to strong binding types, it is not just enough to incorporate into the structure those sequence elements suspected to be responsible for recognition and interaction of chaperones with their natural ligands. Presumably, additional information intrinsic to the conformation of folding intermediates, or even the folding process of the protein itself, is needed. The specific interaction of only the translocated mitochondrial member of the AspAT isozyme system may have evolved to slow down the folding of the protein and hence give it the opportunity to be delivered to the mitochondrial import machinery in a suitable conformational state. By contrast, the cytosolic form, which remains in the compartment where it is synthesized, can skip such interactions and quickly acquire its native conformation. Further investigation is needed to explore the relative rate of cotranslational folding of the two isozymes and the nature of the complexes assembled around the mitochondrial AspAT nascent chain as it is being synthesized on the ribosome.