©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of Mitochondrial Presequences to Yeast Cytosolic Heat Shock Protein 70 Depends on the Amphiphilicity of the Presequence (*)

(Received for publication, October 6, 1995)

Toshiya Endo Satoko Mitsui Masato Nakai David Roise (1)(§)

From the Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan and the Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0506

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interactions between a yeast cytosolic hsp70, Ssa1p, and various synthetic peptides, including mitochondrial presequences, have been studied. The interactions were monitored both indirectly, by measuring the effects of the presequences on the ATPase activity and oligomeric state of the enzyme, and directly, by measuring the increased steady-state fluorescence polarization of fluorescent derivatives of the presequences as they bind to Ssa1p. The presequences are all able to convert Ssa1p from an oligomeric to a monomeric form in a concentration-dependent manner. The presequences are also able to stimulate the ATPase activity of the enzyme at similar concentrations. Quantification of the binding by fluorescence polarization showed that the affinity for Ssa1p is directly related to the physical properties of the presequences. The most amphiphilic presequences, as measured by retention times on reversed-phase high pressure liquid chromatography or surface activity in lipid monolayers, had the highest affinity for Ssa1p. The least amphiphilic presequences, which had previously been shown to be ineffective as mitochondrial targeting sequences, had relatively low affinity for Ssa1p. The results show that Ssa1p interacts with a broad range of amino acid sequences and that the strength of these interactions is related to the physical properties of the sequence. That the physical properties recognized by Ssa1p are identical to those necessary for the targeting function of mitochondrial presequences suggests that Ssa1p may interact with mitochondrial precursor proteins in the cytosol. The interactions may serve a variety of purposes: the maintenance of precursors in translocation-competent forms, the prevention of improper association of precursors with non-mitochondrial membranes, and the delivery of precursors to the mitochondrial surface.


INTRODUCTION

Most nuclear encoded mitochondrial precursor proteins are synthesized on cytosolic ribosomes with amino-terminal presequences that are characterized by positive charge and amphiphilicity. The post-translational targeting of mitochondrial precursors to mitochondria is often facilitated by cytosolic chaperones, including members of the hsp70 stress protein family(1, 2) , mitochondrial import stimulating factor(3, 4) , and presequence binding factor(5, 6) . Mitochondrial precursor proteins are thought to be susceptible to aggregation, and the cytosolic chaperones presumably prevent this undesirable event by binding to the precursors and maintaining the precursors in an import-competent state.

hsp70 proteins function as molecular chaperones by binding to unfolded proteins and releasing the bound proteins at the expense of ATP binding and hydrolysis. The mechanism of recognition of unfolded proteins by hsp70 has been studied mainly by using synthetic peptides(7, 8, 9, 10, 11) . Peptides bind to hsp70 with varying affinity, but no correlation has been found between the amino acid sequence of a peptide and the affinity of the peptide for hsp70(8, 9) . Analysis of a family of peptides with random sequences that bind to BiP, an hsp70 of the endoplasmic reticulum, showed that BiP preferentially binds to peptides containing a cluster of hydrophobic residues(8, 9) . Peptides with high affinity for BiP or for DnaK, a bacterial hsp70, show a preponderance of positively charged residues and relatively few negatively charged residues(10, 11) . On the basis of these results, it has been assumed that cytosolic hsp70 binds to unfolded regions of the mature part of the precursor proteins; these regions probably expose hydrophobic patches that are otherwise buried in the native structure. In the absence of hsp70, the exposed patches would promote aggregation of the proteins.

We have recently found that mitochondrial presequences can induce the aggregation of unfolded proteins and have suggested that mitochondrial precursor proteins may suffer similar consequences if the presequence interacts with the mature part of a precursor during the folding process(12) . The binding of cytosolic chaperones to the presequence of mitochondrial precursor proteins could mask undesirable interactions between the presequence and vulnerable proteins and thereby prevent the aggregation. Presequence binding factor and mitochondrial import stimulating factor may inhibit the aggregation of precursors by similar mechanisms. In the present study, we have found that peptides with amino acid compositions typical for mitochondrial presequences can bind to a yeast cytosolic hsp70, Ssa1p, and can stimulate the ATPase activity of this enzyme. The affinities of these peptides for Ssa1p have been quantified by a novel assay that follows the increased steady-state fluorescence polarization of fluorescein-labeled derivatives of the peptides as the peptides bind to Ssa1p under equilibrium conditions. The affinity of Ssa1p for the peptides is directly related to the amphiphilicity of the peptides, a property that is essential for the targeting function of mitochondrial presequences. Taken together, the results of this work suggest that the interaction of Ssa1p with mitochondrial precursors in the cytosol may serve several functions; it may protect the presequence from improper interactions with itself or other proteins and thus prevent protein aggregation; it may protect the presequence from improper interactions with non-mitochondrial membranes and thus prevent the mistargeting of precursors to other organelles; and it may facilitate the appropriate interactions of presequences with the mitochondrial surface and thus be important in the normal import process.


MATERIALS AND METHODS

Synthetic Peptides

The synthetic peptides used in this study are listed in Fig. 1. The synthesis and characterization of most of the peptides were described previously(13) . WT-CoxIV, Delta11,12-CoxIV, SynA2, SynB2, and SynC were purified by reversed-phase HPLC (^1)on a C(18) column (Vydac, 25 times 0.46 cm) in 0.2% trifluoroacetic acid with a gradient of acetonitrile. Peptide concentrations were determined by amino acid analysis. SCC-(217-232) and ER23 were donated by Dr. K. Mihara (Kyushu University) and Dr. K. Wakamatsu (Gunma University), respectively.


Figure 1: Amino acid sequences of the synthetic peptides. WT-CoxIV, wild-type presequence of yeast cytochrome oxidase subunit IV; Delta11,12-CoxIV, deletion mutant lacking residues 11 and 12 of WT-CoxIV; SynA2, SynB2, SynC, artificial presequences described in (13) ; ER23, a peptide derived from turkey beta receptor protein; SCC(217-232), residues 217-232 of cytochrome P-450.



The CoxIV peptides were labeled at the cysteine residue with 5iodoacetamidofluorescein (Molecular Probes) as described previously(14) . The labeled peptides were purified by reversed-phase HPLC. The Syn peptides were labeled at their amino termini with fluorescein as follows. The pure peptides (0.1 µmol) were dissolved in 25 mM Hepes-NaOH pH 7.4 (0.5 ml) and treated with an equal volume of 0.5 mM 5-carboxyfluorescein, succinimidyl ester (Molecular Probes) that had been dissolved in the same solution. After reaction overnight at room temperature in the dark, the samples were dried under vacuum. The residues were dissolved in the minimal volume of N,N-dimethylformamide and purified by reversed-phase HPLC on a C(18) column. After purification, all the peptides were dried and redissolved in 50% EtOH. The concentration of each of the stock solutions was determined by the absorbance of aliquots diluted into an aqueous buffered solution at pH 8.0 ( = 75,000 M cm). The incorporation of a single fluorescein into each peptide was confirmed by electrospray mass spectrometry at the Scripps Research Institute Mass Spectrometry Facility. The molecular masses ((M + H)) of the labeled peptides were 3376 units for WT-CoxIV (calculated, 3377 units), 3104 units for Delta11,12-CoxIV (calculated, 3101 units), 3131 units for SynA2 (calculated, 3132 units), 3250 units for SynB2 (calculated, 3252 units), and 3130 units for SynC (calculated, 3132 units). All of the fluorescein-labeled Syn peptide samples also contained significant amounts of an oxidized form of the peptides. The covalent modification of the amino terminus presumably increases the sensitivity of the methionine to S-oxidation. The modification does not appear to affect the interactions of the peptides with Ssa1p.

Preparation of hsp70

Ssa1p, a yeast cytosolic hsp70, was purified from Saccharomyces cerevisiae strain Y168 (MATalpha, his-11, 15, leu2-3, 112, lys3-Delta1, Deltatrp1, ura3-52, ssa1::HIS3, ssa2::LEU2, ssa4::LYS2, [pGal:SSA1(URA3)]) according to the previously described procedures (2, 15) with minor modifications. Protein concentrations were determined by the Bradford method (Bio-Rad) and by comparison of SDS-polyacrylamide gel electrophoresis stained with Coomassie Brilliant Blue.

In the binding experiments with fluorescein-labeled presequences, the Ssa1p contained a histidine tag at its carboxyl terminus. This protein was generated by modification of the gene coding for Ssa1p as follows. A NotI restriction site was introduced at the 3`-end of the SSA1 gene, and the (His)(6)-coding sequence, followed by a stop codon, was inserted into this site. The resulting modified SSA1 gene encodes Ssa1p fused to the additional COOH-terminal segment, Ser-Gly-Arg-(His)(6). The expression plasmid pGAP:SSA1-His6 (TRP1), bearing the modified SSA1 gene under the control of GAP promoter, was introduced into the yeast strain, HFSA-11 (MATalpha, his3-11, 15 lys2 ura3 leu2-3, 112 trp1 ssa1::HIS3 ssa2::LEU2 ssa4::LYS2). These cells were grown at 30 °C on YPD medium (2% yeast extract, 1% peptone, 1% dextrose) to late log phase (A = 1.5-2.0). After harvesting and washing, the cells were broken by treatment with Zymolyase-20T (ICN Biomedicals, Inc.) and Dounce homogenization. After sedimentation of the cellular debris, the protein was purified from the soluble cellular extract on a Ni chelate column (Novagen) using the procedure supplied by the manufacturer. The affinity-purified protein was further fractionated by cation-exchange chromatography on a MonoS column (Pharmacia Biotech Inc.) using conditions similar to those described by Chirico et al.(15) . Concentration of the protein was determined by absorbance using an extinction coefficient ( = 35,700 M cm) obtained from quantitative amino acid analysis.

Preparation of Reduced and Carboxymethylated Lactalbumin (RCM-LA) and Apocytochrome c

RCM-LA was prepared from bovine alpha-lactalbumin (Sigma L-6010) as described previously(12) . Apocytochrome c was prepared from horse heart cytochrome c (Sigma C-7752) as described previously(16, 17) . Concentrations of apocytochrome c solutions were determined spectrophotometrically using an extinction coefficient at 280 nm of 12,500 M cm.

Gel Filtration Experiments

Ssa1p and ligands (RCM-LA or synthetic peptides) were incubated in 20 mM Hepes-KOH, pH 7.4, 10 mM MgCl(2), and 5 mM nucleotide (ADP, ATP, ATPS, or AMPPNP) in a volume of 30 µl for 1 h at 37 °C. The mixtures were passed through 0.20-µm filters and applied to a Superose 12 column that had been equilibrated with 20 mM Hepes-KOH, pH 7.4 and 150 mM KCl. The column was eluted with the same buffered solution at 10 °C using a SMART system (Pharmacia). Elution profiles were monitored at 280 nm and processed with the software, SMART MANAGER, supplied by Pharmacia.

Measurement of ATPase Activity of Ssa1p

Ssa1p (0.47 µM; Ssa1p is monomeric at this concentration) was incubated with various concentrations of ligands (proteins or peptides) in 20 mM Hepes-KOH, pH 7.4, 50 mM KCl, 10 mM MgCl(2), and 100 µM [^3H]ATP (0.2 Ci mmol) in a volume of 10 µl at 30 °C for 1 h. Samples were placed on ice, and aliquots (1 µl) were analyzed for ADP by thin-layer chromatography on polyethyleneimine cellulose(7) . The rates of ATP hydrolysis were plotted against ligand concentration after subtraction of the rate of ATP hydrolysis in the absence of ligand. The data (averages of at least four independent experiments) were fit to the equation,

where v is the net rate of hydrolysis of ATP after subtraction of the rate in the absence of added peptide or protein, [P] is the concentration of the peptide or protein ligand, V is a constant corresponding to the maximal net activated rate of hydrolysis of ATP, and K is a constant corresponding to the concentration of ligand that is required for the half-maximal net rate of hydrolysis of ATP. In the cases where high concentrations of peptides or proteins caused a decrease in the rate of hydrolysis of ATP, the rates at the highest concentrations were not included in the curve fitting.

Binding Assays with Fluorescein-labeled Presequences

Various amounts of the histidine-tagged Ssa1p were diluted from a concentrated stock solution into 20 mM Hepes-KOH, pH 7.4, 100 mM KCl, 5 mM MgCl(2), and 1 mM ADP or ATP (200 µl, final). The fluorescent peptides were added from concentrated stock solutions in 50% EtOH (25-100 nM, final). The samples were incubated at 30 °C for 30 min, and the steady-state anisotropy of each sample was measured on an SLM-Aminco SPF-500C spectrofluorometer (excitation, 490 nm; emission, 525 nm; 5-nm bandwidths) using the polarization option supplied by the manufacturer. The anisotropy, r, is described by the equation,

where I corresponds to the fluorescence intensity measured when the excitation and emission polarizers are in the vertical position, and I is the intensity with the excitation polarizer in the vertical position and the emission polarizer in the horizontal position(18) . The instrument correction factor, G, is defined as II, with I equal to the fluorescence intensity when the excitation polarizer is horizontal and the emission polarizer is vertical, and I is the intensity when both polarizers are horizontal. The value of G was constant for all fluorescein-containing samples at a given instrument setting. Each reported anisotropy reflects the average value obtained from five measurements each of I and I.


RESULTS

Effects of Presequences on the Oligomeric State of Ssa1p

We have examined the interactions between synthetic presequences and purified yeast cytosolic hsp70, Ssa1p, by three independent approaches: 1) gel filtration chromatography to monitor the effects of presequences on the oligomeric state of Ssa1p; 2) ATPase assays to follow the effects of presequences on the enzyme activity of Ssa1p; and 3) binding assays that measure the affinity of presequences for Ssa1p. In each case, a variety of synthetic peptides have been compared (Fig. 1); the structural features, interactions with lipid membranes, and abilities to target proteins to mitochondria have been studied in detail for most of these sequences(13) .

Gel filtration chromatography of Ssa1p in the presence of Mg-ADP showed that in the absence of unfolded proteins or peptides, Ssa1p exists in multiple forms (Fig. 2A). The major form, which eluted at 30 min in our SMART system, corresponds to the monomer. Two other forms, which eluted at 27.5 and 26 min, probably correspond to the dimer and trimer, respectively. As the concentration of Ssa1p was lowered, the peaks due to the dimer and trimer decreased and that of the monomeric form increased concomitantly (not shown). The dimer and trimer were also dissociated into the monomeric form by incubation with Mg-ATP but not by incubation with the nonhydrolyzable ATP analogues, Mg-AMPPNP or Mg-ATPS (Fig. 2A). The slight difference in the elution volumes of the Ssa1p monomer in the samples containing Mg-ATP and in those containing Mg-ADP may reflect a dynamic equilibrium between the monomer and the multimers in the presence of Mg-ADP. The dependence of the oligomeric state of Ssa1p on nucleotides suggests that Ssa1p may associate with itself by a mechanism similar to that occurring when it binds to unfolded proteins.


Figure 2: Gel filtration chromatography of complexes formed between RCM-LA and Ssa1p. Ssa1p (2.5 µM) was incubated with ADP, ATPS, AMPPNP, or ATP (5 mM) in the absence (A) or presence (B) of RCM-LA (2.5 µM) at 37 °C for 1 h and subjected to gel filtration on Superose 12.



Ssa1p and a model unfolded protein, RCM-LA, formed a stable complex during incubation in the presence of Mg-ADP at 37 °C for 1 h (Fig. 2B). The complex was observed as a peak with an elution volume similar to that of the Ssa1p dimer. It could be distinguished from the Ssa1p dimer by changing the concentration of Ssa1p (data not shown). The presence of Mg-ATP, but not of the nonhydrolyzable ATP analogues, inhibited the formation of the Ssa1p-RCM-LA complex (Fig. 2B), and complexes formed during incubation with Mg-ADP could subsequently be dissociated by the addition of excess Mg-ATP to the solution (not shown).

Incubation of the wild-type yeast cytochrome oxidase subunit IV presequence (WT-CoxIV) with Ssa1p in the presence of Mg-ADP resulted in a decrease in the amounts of the Ssa1p dimer and trimer and a concomitant increase in a form that eluted slightly earlier than the Ssa1p monomer (Fig. 3A, WT-CoxIV + ADP). For comparison, the presequence had no effect on the migration of the monomeric form of Ssa1p that predominates in the presence of Mg-ATP (Fig. 3A, WT-CoxIV + ATP). These results suggest that the wild-type presequence forms a complex with Ssa1p in the presence of Mg-ADP but not in the presence of Mg-ATP and that the formation of the Ssa1p-WT-CoxIV complex competes with the formation of Ssa1p oligomers. The presequence and RCM-LA therefore bind to Ssa1p in a similar manner. The effect of the presequence on the self-association of Ssa1p could be used as an assay to monitor the interactions between the presequence and Ssa1p. The decrease in self-association was found to depend strongly on the concentration of the WT-CoxIV (Fig. 4).


Figure 3: Gel filtration chromatography of complexes formed between Ssa1p and WT-CoxIV or SynB2. Ssa1p (1 µM) was incubated with ADP or ATP (5 mM) in the absence or presence of 4 µM WT-CoxIV (A) or 4 µM SynB2 (B) at 37 °C for 1 h as indicated. Samples were then subjected to gel filtration on Superose 12.




Figure 4: Competition between self-association of Ssa1p and peptide binding to Ssa1p. Ssa1p (1 µM) was incubated with ADP (5 mM) in the presence of various synthetic peptides at 37 °C for 1 h. The samples were then subjected to gel filtration on Superose 12. The amounts of Ssa1p present as oligomers at this time are plotted as a function of the concentrations of peptides. The self-association of Ssa1p that occurs in the absence of peptide was arbitrarily set to 100%.



In order to understand what features of the presequence promote the association with Ssa1p, we compared the effects of various presequence analogues on the aggregation of Ssa1p. WT-CoxIV, SynA2, SynB2, SynC, and Delta11,12-CoxIV have 5 or 6 positive charges, have no negative charges, are rich in serine residues, and are similar in length (Fig. 1). However, WT-CoxIV, SynA2, SynC, and Delta11,12-CoxIV can, when attached to passenger proteins, target the proteins to mitochondria, whereas SynB2 cannot(13, 19) . The relative amphiphilicity of the synthetic presequences is SynA2 > SynC > WT-CoxIV > Delta11,12-CoxIV > SynB2, and this property correlates with the ability of the presequences to target a passenger protein to mitochondria(13) .

With the exception of SynA2, the presequences were found to inhibit the formation of Ssa1p oligomers to an extent that paralleled the amphiphilicity of the presequences (Fig. 4). It is not particularly surprising that the behavior of the SynA2 presequence is somewhat unusual in these assays, since this peptide was found to have a significant amount of beta-structure, even in the absence of detergent micelles(13) . The presence of secondary structure in aqueous solution suggests that SynA2 may be aggregated at the relatively high concentrations used to inhibit the formation of Ssa1p oligomers and that the effective concentration of the monomeric form of SynA2 is somewhat lower than the total concentration. A more surprising result was the observation that the SynB2 presequence had a significant inhibitory effect on the aggregation of Ssa1p. Previous studies showed that BiP, the hsp70 of the lumenal compartment of the endoplasmic reticulum, preferentially binds to peptides containing hydrophobic residues or a subset of aromatic and hydrophobic amino acids in alternating positions(8, 9) . Although WT-CoxIV, Delta11,12-CoxIV, SynA2, and SynC contain several hydrophobic amino acid residues that are separated by 1-3 residues and that may provide binding sites for Ssa1p, SynB2 has hydrophobic amino acid residues only at the ends (ML at the NH(2) terminus and YLL at the COOH terminus) and would not have been expected to bind to Ssa1p. The interaction of SynB2 with Ssa1p was found to exhibit the same dependence on nucleotides as the WT-CoxIV presequence; SynB2 associated with Ssa1p in the presence of Mg-ADP but not in the presence of Mg-ATP (Fig. 3B). Since the minimum length for tight binding of peptides to hsp70 is 7-8 amino acid residues(8) , it is unlikely that SynB2 only binds to Ssa1p through the short segments of hydrophobic amino acid residues on its ends. Other features must, therefore, be responsible for the interactions of SynB2 with Ssa1p. As a control, we confirmed that Ac-ER23, which contains two negatively charged amino acids in proximity to four positively charged amino acids (Fig. 1), has a much weaker effect on the oligomeric state of Ssa1p than the other peptides (Fig. 4).

Stimulation of the ATPase Activity of Ssa1p by Presequences

Since it has been reported that the binding of some, but not all, short peptides or unfolded proteins to BiP and to mammalian cytosolic hsp70 stimulates the ATPase activity of hsp70 (7, 8, 9, 20, 21) , we next examined the effects of the mitochondrial presequences on the ATPase activity of Ssa1p. We first tested the effects of two unfolded proteins, apocytochrome c and RCM-LA, on the ATPase activity of Ssa1p. Addition of apocytochrome c or RCM-LA enhanced the ATPase activity of Ssa1p (Fig. 5A); the concentrations of apocytochrome c and RCM-LA required for half-maximal activation (K) are 3.4 and 9.6 µM, respectively.


Figure 5: Stimulation of the ATPase activity of Ssa1p by peptides and unfolded proteins. Ssa1p (0.47 µM) was incubated with [^3H]ATP (100 µM) and the indicated concentrations of peptides or unfolded proteins at 30 °C for 1 h. The formation of ADP was assayed by thin-layer chromatography. The rates were plotted as a function of the concentrations of peptide or protein after subtraction of background formation of ADP that occurs in the absence of peptide or protein. The curves are drawn according to the equation given in the text.



WT-CoxIV and SynB2 also stimulated the ATPase activity of Ssa1p, with K values of 5.6 and 9.7 µM, respectively (Fig. 5B). As with the unfolded proteins, the stimulation decreased at higher concentrations of WT-CoxIV (>20 µM), due perhaps to the aggregation of the presequence or to damaging effects of the surface-active presequence on the structure and activity of Ssa1p. We confirmed that SCC-(217-232), a control peptide that contains several acidic amino acids (Fig. 1), shows little effect on the ATPase activity of Ssa1p (Fig. 5B). These results, together with those of the gel filtration chromatography, demonstrate that model mitochondrial presequences and unfolded proteins can interact with Ssa1p in a similar manner.

Direct Measurement of the Binding of Presequences to Ssa1p

The results described above provided indirect evidence that mitochondrial presequences can bind to Ssa1p. In order to demonstrate the binding directly and to quantify the affinity of the presequences for Ssa1p, we developed a new approach using steady-state fluorescence polarization to assess the binding. Fluorescein-labeled derivatives of WT-CoxIV and Delta11,12-CoxIV had been used previously to analyze the binding and import of presequences into isolated yeast mitochondria (14, 22) . In those experiments, the steady-state fluorescence of the labeled presequences is quenched as the presequences bind to the surface of the mitochondria, and the quenching can be used directly to calculate binding constants for the presequences. In the case of binding to Ssa1p, there is no change in the fluorescence intensity of the fluorescein-labeled WT-CoxIV presequence in the presence of Ssa1p, but the anisotropy of the solution increases significantly. Since changes in anisotropy are directly related to changes in the fraction of a fluorescent molecule present in a larger complex(18) , this approach can be used to calculate equilibrium dissociation constants for the binding interaction.

In order to analyze the binding of the Syn presequences to Ssa1p by changes in fluorescence anisotropy, a fluorescein probe had to be incorporated into these molecules as well. The Syn presequences lack cysteine residues, however, so iodoacetamido-linked derivatives of fluorescein could not be used for this purpose. Instead, we took advantage of the reactivity of the amino termini of the peptides and used a succinimidyl ester of 5-carboxyfluorescein for the labeling reactions. We felt that labeling at this position would also minimize the effects of the label on the interactions of the labeled peptides with Ssa1p. The reactions proceeded smoothly, and pure, fluorescein-labeled peptides were obtained in good yield after purification by reversed-phase HPLC.

The steady-state fluorescence anisotropy of the five labeled presequences at low concentrations (25-100 nM) in the absence of Ssa1p was between 0.055 and 0.075. For comparison, the anisotropy of the fluorescein labeling reagents alone was approximately 0.04. The values of anisotropy are consistent with the relative sizes of the respective molecules and suggest that the labeled presequences exist as monomers under these conditions. In the presence of Mg-ADP and increasing concentrations of Ssa1p, the anisotropy of the presequences was found to increase dramatically (Fig. 6A), while that of free fluorescein was unaffected by the presence of Ssa1p. The increase in anisotropy of the bound presequences results from the lower rotational diffusion of the fluorophore in the bound complexes compared with that in the free presequences. These observations confirm that the presequences are able to bind to Ssa1p and demonstrate that the fluorophore alone does not.


Figure 6: Binding of the fluorescein-labeled presequences to Ssa1p. The peptides (25-100 nM) were incubated with the indicated concentrations of Ssa1p in the presence of 1 mM ADP (A) or 1 mM ATP (B) for 30 min at 30 °C. The fluorescence anisotropy of the solutions was measured, and the data were fit to . The parameters obtained from the curve fitting (Table 1) were used to generate the lines shown for each peptide: , SynA2; bullet, SynC; , WT-CoxIV; , Delta11,12-CoxIV; and bullet, SynB2.





At low concentrations of presequences, the increases in anisotropy of the fluorescence in the presence of Ssa1p are well described by a hyperbolic function of the form,

where r is the measured anisotropy, [Ssa1p] is the free concentration of Ssa1p, r and r are constants corresponding to the anisotropy of the free and bound forms of the presequence, respectively, and K(d) is the dissociation constant for the complex between Ssa1p and the presequence. For most of the experiments, the concentration of the presequence was much lower than the value of K(d), and it was assumed that [Ssa1p] was equal to the total concentration of Ssa1p. This assumption is consistent with the observation that under the conditions used in the binding assays, the measured anisotropies are relatively independent of the exact concentration of peptide used. The independence of the anisotropy on the concentration of presequence is convenient, given the stickiness of the presequences and the difficulty in obtaining identical concentrations of a presequence in each binding assay.

Plots of the anisotropy of the labeled presequences as a function of the concentration of Ssa1p in the presence of 1 mM Mg-ADP show that the affinities of the peptides for Ssa1p vary in the order SynA2 SynC > WT-CoxIV > Delta11,12-CoxIV > SynB2 (Fig. 6A). In curve fitting of the data from the binding experiments with SynA2 and SynC to , it was possible to obtain good fits for the parameters r, r, and K(d) (Table 1). Although concentrations of Ssa1p sufficient to saturate the binding could not be reached for the other presequences, values of K(d) could still be estimated for those presequences by assigning a value of r equal to 0.25, the average of the values of r obtained from the experiments with SynA2 and SynC. The good overlap between the lines generated from the parameters obtained by curve fitting (Table 1) and the data points (Fig. 6) argues that the use of a single value of r for all the presequences is appropriate. The small increase in anisotropy of SynB2 indicates that this relatively hydrophilic presequence can indeed bind to Ssa1p and confirms the effects of SynB2 on the oligomeric state and ATPase activity of the protein.

Substitution of Mg-ADP with Mg-ATP in the binding assays caused a significant decrease in the affinity of Ssa1p for the presequences (Fig. 6B). In order to calculate values of K(d), it was necessary to assume that the value of r remains the same as in the experiments with ADP. Because of the low affinities of Delta11,12-CoxIV and SynB2, it was not possible to calculate dissociation constants for these presequences in the presence of Mg-ATP. For the other presequences, however, the presence of ATP was found to decrease the affinities by roughly 4-fold (Table 1). The effect of Mg-ATP on the binding is consistent with the effects of Mg-ATP that were observed on the migration behavior of Ssa1p in gel filtration chromatography.


DISCUSSION

In the present study, we have examined the interactions between mitochondrial presequences and the yeast cytosolic hsp70, Ssa1p. The results demonstrate that the presequences form stable complexes with Ssa1p in the presence of Mg-ADP or non-hydrolyzable ATP analogues and that the presence of Mg-ATP significantly weakens these interactions. In addition, the mitochondrial presequences were found to stimulate the ATPase activity of Ssa1p with kinetic parameters similar to those observed with the unfolded proteins, apocytochrome c and RCM-LA.

The affinities of the presequences for Ssa1p measured here are considerably higher than those typically reported for the binding of other peptides to hsp70s(7, 9, 23) . The higher affinities may be due to several factors. First, Ssa1p may simply bind peptides more strongly than do the other related members of the hsp70 family. Indeed, the affinity of Ssa1p for a fragment of cytochrome c was found to be severalfold higher than that of bovine hsp70(23) . Other direct comparisons between the binding affinity of Ssa1p and other hsp70s have not yet been made. Second, the high affinities observed here may be due to the method used to measure the binding. The assays typically used to follow this process require the separation of free and bound forms of the labeled peptide, either by rapid gel filtration(7, 11, 23) , precipitation with trichloroacetic acid and filtration(9) , or native gel electrophoresis(10) . These methods are likely to underestimate the true binding affinities, particularly of weaker interactions, because of partial dissociation of the complexes during the separation step. In the approach used here, binding is observed directly in solution, under equilibrium conditions, and a separation step is not necessary. The dissociation constants obtained in these experiments are, therefore, more likely to reflect the true affinities. Finally, the higher affinities reported here for the presequences may be due to the length of these peptides. Previous work has focused on the binding of relatively short peptides to hsp70s, since it was found that the stimulation of the ATPase activity of BiP by random peptide libraries did not increase as the peptides were lengthened beyond 7 residues(8) . A similar dependence on length was found in competition assays with radiolabeled probes. These results suggest that peptides containing at least seven amino acids are favored for binding, but they do not exclude the possibility that certain longer peptides may interact better. Although we have not examined the effects of peptide length on binding in the current work, a recent study found that the affinity of a 21-residue peptide for DnaK was 20-fold higher than that of a peptide composed of only the amino-terminal 7 residues of the same sequence (24) . In those experiments, which were measured under equilibrium conditions using a solution phase, fluorescence assay, the K(d) of the longer peptide was 0.063 µM in the absence of added nucleotide and 2.2 µM in the presence of Mg-ATP. Thus, the affinity of this peptide for DnaK is fairly similar to that observed with SynA2 and SynC in our experiments with Ssa1p, although the effect of ATP on the affinity of DnaK is somewhat larger.

Our most intriguing finding is that the interactions of mitochondrial presequences with Ssa1p are directly correlated with the amphiphilicity of the presequences (Table 1). The presequences had previously been shown to insert into monolayers of negatively charged phospholipids in the relative order SynA2 > SynC > WT-CoxIV > Delta11,12-CoxIV > SynB2 (13) . The same relative ranking was found for the retention of the presequences on reversed-phase HPLC (Table 1). That the binding affinities of the presequences for Ssa1p mirror these physical properties suggests that the binding to hsp70s may depend less on the specific primary sequence of a peptide than on the higher level physical properties of the peptide. Indeed, the SynA2 and SynC presequences, which are isomeric and which differ only in primary sequence, display remarkably similar surface activities and retention on HPLC, yet have very different circular dichroism spectra(13) . The nearly identical binding of these peptides to Ssa1p argues that structure and primary sequence are less important for the binding than is amphiphilicity. The flexibility in the structural requirements for binding to Ssa1p may be related to the finding that different chaperones can bind the same peptide in different conformations(25) . It is also interesting to note that GroEL, a bacterial chaperonin, has been shown by NMR to bind an amphipathic alpha-helical peptide that is part of the mitochondrial targeting sequence of rhodanese(26) .

The ability of SynB2 to bind to Ssa1p was somewhat of a surprise. This sequence, which contains hydrophobic residues only at its ends, required only slightly higher concentrations than WT-CoxIV to stimulate the ATPase activity of Ssa1p (Fig. 5) and was found to bind to Ssa1p weakly in the presence of ADP (Fig. 6A). Since the SynB2 presequence is not capable of transporting attached passenger proteins into mitochondria(13, 19) , the binding of SynB2 by Ssa1p argues against a role of Ssa1p in determining the specificity of targeting of precursors to mitochondria. The result does not, however, preclude the involvement of Ssa1p at another step in the process.

We have recently suggested that mitochondrial presequences, which are characterized by positive charge and amphiphilicity, can induce aggregation of mitochondrial precursor proteins during the folding process(12) . Taken together with the present findings that cytosolic hsp70 can bind to mitochondrial presequences, the role of cytosolic hsp70 in the prevention of precursor aggregation may need to be broadened. Not only can hsp70 prevent aggregation by binding to the unfolded, mature part of a mitochondrial precursor protein, but it may also prevent aggregation by binding to the presequence itself. While associated with hsp70, the presequence would be prevented from interacting with the newly synthesized, unfolded part of the precursor and would not be able to induce aggregation of these proteins in the cytosol. The protective effects of chaperones may also extend to the prevention of improper association of precursor proteins with non-mitochondrial membranes. The high affinity of Ssa1p for the presequences with highest surface activity suggests that Ssa1p could sequester the presequences in the cytosol until it is appropriate for them to interact with the mitochondrial surface.


FOOTNOTES

*
This work was supported in part by a grant for the ``Biodesign Research Program'' from The Institute of Physical and Chemical Research (RIKEN), Grants-in-aid for Scientific Research 05454621 and 04259101 from the Ministry of Education, Science and Culture of Japan, grants from the Toray Science Foundation, the Ciba-Geigy Foundation, and the Daiko Foundation (to T. E.), and National Science Foundation Grant MCB-9316810 (to D. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Palo Alto Inst. of Molecular Medicine, 2462 Wyandotte St., Mountain View, CA 94043. Tel.: 415-694-1420; Fax: 415-694-7717; :droise{at}hooked.net.

(^1)
The abbreviations used are: HPLC, high pressure liquid chromatography; RCM-LA, reduced carboxymethylated bovine alpha-lactalbumin; ATPS, adenosine 5`-O-3-thiotriphosphate; AMPPNP, adenosine 5`-(beta,-imino)triphosphate.


ACKNOWLEDGEMENTS

We thank Dr. K. Mihara (Kyushu University) and Dr. K. Wakamatsu (Gunma University) for the peptides SCC-(217-232) and ER23, respectively, Dr. Elizabeth Craig for the SSA1 gene, Dr. Merritt Maduke for amino acid analysis, and Osama Khouri and Paul Sigala for comments on the manuscript.


REFERENCES

  1. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A., and Schekman, R. (1988) Nature 332, 800-805 [CrossRef][Medline] [Order article via Infotrieve]
  2. Murakami, H., Pain, D., and Blobel, G. (1988) J. Cell Biol. 107, 2051-2057 [Abstract]
  3. Hachiya, N., Alam, R., Sakasegawa, Y., Sakaguchi, M., Mihara, K., and Omura, T. (1993) EMBO J. 12, 1579-1586 [Abstract]
  4. Hachiya, N., Komiya, T., Alam, R., Iwahashi, J., Sakaguchi, M., Omura, T., and Mihara, K. (1994) EMBO J. 13, 5146-5154 [Abstract]
  5. Murakami, K., and Mori, M. (1990) EMBO J. 9, 3201-3208 [Abstract]
  6. Murakami, K., Tanase, S., Morino, Y., and Mori, M. (1992) J. Biol. Chem. 267, 13119-13122 [Abstract/Free Full Text]
  7. Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390 [Medline] [Order article via Infotrieve]
  8. Flynn, G. C., Pohl, J., Flocco, M. T., and Rothman, J. E. (1991) Nature 353, 726-730 [CrossRef][Medline] [Order article via Infotrieve]
  9. Blond-Elguindi, S., Cwirla, S. E., Dower, W. J., Lipshutz, R. J., Sprang, S. R., Sambrook, J. F., and Gething, M. J. (1993) Cell 75, 717-728 [Medline] [Order article via Infotrieve]
  10. Fourie, A. M., Sambrook, J. F., and Gething, M. J. (1994) J. Biol. Chem. 269, 30470-30478 [Abstract/Free Full Text]
  11. Gragerov, A., Zeng, L., Zhao, X., Burkholder, W., and Gottesman, M. E. (1994) J. Mol. Biol. 235, 848-854 [CrossRef][Medline] [Order article via Infotrieve]
  12. Endo, T., Mitsui, S., and Roise, D. (1995) FEBS Lett. 359, 93-96 [CrossRef][Medline] [Order article via Infotrieve]
  13. Roise, D., Theiler, F., Horvath, S. J., Tomich, J. M., Richards, J. H., Allison, D. S., and Schatz, G. (1988) EMBO J. 7, 649-653 [Abstract]
  14. Roise, D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 608-612 [Abstract]
  15. Chirico, W. J., Waters, M. G., and Blobel, G. (1988) Nature 332, 805-810 [CrossRef][Medline] [Order article via Infotrieve]
  16. Fisher, W. R., Taniuchi, H., and Anfinsen, C. B. (1973) J. Biol. Chem. 248, 3188-3195 [Abstract/Free Full Text]
  17. Hamada, D., Hoshino, M., Kataoka, M., Fink, A. L., and Goto, Y. (1993) Biochemistry 32, 10351-10358 [Medline] [Order article via Infotrieve]
  18. Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy , 1st Ed., pp. 125-131, Plenum Press, New York
  19. Allison, D. S., and Schatz, G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9011-9015 [Abstract]
  20. Palleros, D. R., Welch, W. J., and Fink, A. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5719-5723 [Abstract]
  21. Sadis, S., and Hightower, L. E. (1992) Biochemistry 31, 9406-9412 [Medline] [Order article via Infotrieve]
  22. Swanson, S. T., and Roise, D. (1992) Biochemistry 31, 5746-5751 [Medline] [Order article via Infotrieve]
  23. Greene, L. E., Zinner, R., Naficy, S., and Eisenberg, E. (1995) J. Biol. Chem. 270, 2967-2973 [Abstract/Free Full Text]
  24. Schmid, D., Baici, A., Gehring, H., and Christen, P. (1994) Science 263, 971-973 [Medline] [Order article via Infotrieve]
  25. Landry, S. J., Jordan, R., McMacken, R., and Gierasch, L. M. (1992) Nature 355, 455-457 [CrossRef][Medline] [Order article via Infotrieve]
  26. Landry, S. J., and Gierasch, L. M. (1991) Biochemistry 30, 7359-7362 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.