Import of a mitochondrial presequence into protein-free phospholipid vesicles.

A synthetic mitochondrial presequence has been shown to translocate across pure phospholipid bilayers. The presequence was fluorescently labeled so that its association with membranes could be monitored spectroscopically. In the presence of large unilamellar vesicles, the presequence showed time- and potential-dependent protection from reaction with added trypsin and dithionite. The protection was rapidly reversed by treatment of the vesicles with detergent. If the vesicles contained trypsin, the added presequence became sensitive to digestion by the protease. The results show that a mitochondrial presequence can translocate across phospholipid bilayers that lack a hydrophilic translocation pore.

Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides.

The bovine heart mitochondrial F1-ATPase is inhibited by a number of amphiphilic cations. The order of effectiveness of non-peptidyl inhibitors examined as assessed by the concentration estimated to produce 50% inhibition (I0.5) of the enzyme at pH 8.0 is: dequalinium (8 microM), rhodamine 6G (10 microM), malachite green (14 microM), rosaniline (15 microM) greater than acridine orange (180 microM) greater than rhodamine 123 (270 microM) greater than rhodamine B (475 microM), coriphosphine (480 microM) greater than safranin O (1140 microM) greater than pyronin Y (1650 microM) greater than Nile blue A (greater than 2000 microM). The ATPase activity was also inhibited by the following cationic, amphiphilic peptides: the bee venom peptide, melittin; a synthetic peptide corresponding to the presence of yeast cytochrome oxidase subunit IV (WT), and amphiphilic, synthetic peptides which have been shown (Roise, D., Franziska, T., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S. and Schatz, G. (1988) EMBO J. 7, 649-653) to function in mitochondrial import when attached to dihydrofolate reductase (delta 11.12, Syn-A2, and Syn-C). The order of effectiveness of the peptide inhibitors as assessed by I0.5 values is: Syn-A2 (40 nM), Syn-C (54 nM) greater than melittin (5 microM) greater than WT (16 microM) greater than delta 11,12 (29 microM). Rhodamines B and 123, dequalinium, melittin, and Syn-A2 showed noncompetitive inhibition, whereas each of the other inhibitors examined (rhodamine 6G, rosaniline, malachite green, coriphosphine, acridine orange, and-Syn-C) showed mixed inhibition. Replots of slopes and intercepts from Lineweaver-Burk plots obtained for dequalinium were hyperbolic indicating partial inhibition. With the exception of Syn-C, for which the slope replot was hyperbolic and the intercept replot was parabolic, steady-state kinetic analyses indicated that inhibition by the other inhibitors was complete. The inhibition constants obtained by steady-state kinetic analyses were in agreement with the I0.5 values estimated for each inhibitor examined. Rhodamine 6G, rosaniline, dequalinium, melittin, Syn-A2, and Syn-C were observed to protect F1 against inactivation by the aziridinium of quinacrine mustard in accord with their experimentally determined I0.5 values.(ABSTRACT TRUNCATED AT 400 WORDS)

Import of a mitochondrial presequence into P. denitrificans. Insight into the evolution of protein transport.

According to the endosymbiont hypothesis, mitochondria are descended from ancient aerobic bacteria that were engulfed by protoeukaryotic cells. Experiments described here show that a synthetic peptide corresponding to a yeast mitochondrial targeting sequence can be imported into Paracoccus denitrificans, a soil bacterium thought to be closely related to the protomitochondrion. The import is very similar to that observed with isolated yeast mitochondria. The results suggest that the protomitochondrion may have been inherently able to translocate mitochondrial presequences. This ability may partly explain the development of the protein import process during the evolution of the mitochondrion.

Mitochondrial presequences can induce aggregation of unfolded proteins.

We have studied the interactions between various synthetic peptides and two model unfolded proteins, reduced alpha-lactalbumin and reduced and carboxymethylated alpha-lactalbumin. We found that mitochondrial presequences could induce aggregation of the unfolded alpha-lactalbumins but not of the native alpha-lactalbumin. The presequence-induced aggregation of unfolded alpha-lactalbumin was dependent on electrostatic interactions and on the amphiphilicity of the presequences. Since positive charge and amphiphilicity are necessary for the targeting function of mitochondrial presequences, presequence-induced aggregation may be responsible for the instability of mitochondrial precursor proteins and may need to be inhibited by binding factors in the cytosol.

N-terminal half of a mitochondrial presequence peptide takes a helical conformation when bound to dodecylphosphocholine micelles: a proton nuclear magnetic resonance study.

Two-dimensional proton nuclear magnetic resonance (NMR) spectra of a synthetic peptide (p25) corresponding to the amino-terminus of the yeast mitochondrial cytochrome oxidase subunit IV precursor protein have been analyzed. Sequence-specific resonance assignments of the peptide have been made in the presence of micelles of a phospholipid analog, perdeuterated dodecylphosphocholine (DPC), with the aid of such techniques as HOHAHA, DQF-COSY, and NOESY. The interresidue nuclear Overhauser effects (NOEs) indicate that the N-terminal half of p25 (S3-F11) takes a helical structure while the C-terminal half does not take a regular secondary structure. Addition of DPC to the solution of p25 induced chemical shift changes only of the resonances from the residues in the N-terminal half, suggesting that the N-terminal half of p25 is directly involved in binding to DPC. The induced helical structure in the N-terminal half at a lipid-water interface may be important in the ability of this presequence to direct a "passenger" protein into mitochondria.

Interaction of a synthetic mitochondrial presequence with isolated yeast mitochondria: mechanism of binding and kinetics of import.

The mechanism of interaction of a presequence with isolated yeast mitochondria was examined. A synthetic peptide corresponding to a matrix-targeting signal was covalently labeled with a fluorescent probe. Binding of the presequence to the surface of the mitochondria and translocation of the presequence into the interior of the mitochondria could then be monitored directly in solution by measuring changes in the steady-state fluorescence of the attached fluorophore. The binding step was rapid and reversible. Quantitation of the binding under equilibrium conditions suggested that the initial association of the presequence with the surface of the mitochondria occurred by partitioning of the presequence directly into the lipid bilayer of the outer membrane. Subsequent translocation of the bound presequence into the mitochondria was monitored by measuring the rate of disappearance of presequences sensitive to digestion by added trypsin. The efficiency of translocation was high, and the rate of the translocation was dependent on the electrical potential across the inner membrane. At physiological concentrations of presequence, the rate displayed first-order kinetics with respect to the concentration of bound presequence and had a rate constant of 0.19 min-1 at 20 degrees C. Several kinetic models for the translocation of the presequence are presented that are consistent with the experimental results.

Recognition and binding of mitochondrial presequences during the import of proteins into mitochondria.

Nuclear-encoded mitochondrial proteins are imported into mitochondria due to the presence of a targeting sequence, the presequence, on their amino termini. Presequences, which are typically proteolyzed after a protein has been imported into a mitochondrion, lack any strictly conserved primary structure but are positively charged and are predicted to form amphiphilic alpha-helices. Studies with synthetic peptides corresponding to various presequences argue that presequences can partition nonspecifically into the mitochondrial outer membrane and that the specificity of translocation of precursors into mitochondria may depend on interactions of the presequence with the electrical potential of the inner membrane. Although proteins of the outer membrane that are necessary for the translocation of precursor proteins have been proposed to function as receptors for presequences, the binding of presequences to these proteins has not been demonstrated directly. Proteins of the mitochondrial outer membrane may not be responsible for the specificity of translocation of precursors but may instead function, together with cytosolic molecular chaperones, to maintain precursor proteins in conformations that are competent for translocation as the precursors associate with the mitochondrial surface.

Binding of a mitochondrial presequence to natural and artificial membranes: role of surface potential.

The binding of a synthetic mitochondrial presequence to large, negatively charged, unilamellar vesicles and to unenergized yeast mitochondria has been measured. The presequence, which corresponds to the amino-terminal 25 residues of the yeast cytochrome oxidase subunit IV precursor, was labeled with a fluorescent probe and used to examine the importance of the surface potentials of membranes on the interactions with the presequence. Binding of the fluorescent presequence to the membranes was determined by measuring a decrease in the fluorescence emission of the bound presequence. Binding both to the vesicles and to the mitochondria could be described as a simple partitioning of the presequence between the aqueous and lipid phases. The partitioning was found to depend on the ionic strength of the medium, and the Gouy-Chapman theory could be used to describe the partitioning at various ionic strengths. Application of the theory allowed the determination of an apparent charge on the presequence (+2.31 +/- 0.25), salt-independent apparent partition coefficients for vesicles (99 +/- 84 M-1) and for unenergized mitochondria (14.5 +/- 3.6 L g-1), and an estimated charge density for the mitochondrial outer membrane (-0.0124 +/- 0.0016 C m-2). This study shows that electrostatic effects are significant for the binding of a mitochondrial presequence both to lipid vesicles and to mitochondria, the natural target membrane of the presequence. The accumulation of positively charged presequences at the negative mitochondrial surface and the subsequent partitioning of the presequences directly into the mitochondrial outer membrane probably represent early steps in the translocation of precursor proteins into mitochondria.

Partitioning of a-factor analogues into membranes: analysis of binding and importance for biological activity.

Analogues of the a-factor mating pheromone of the yeast Saccharomyces cerevisiae were used to measure interactions of the pheromones with lipid vesicles and with isolated yeast membranes. The binding of the analogues of a-factor to vesicles and to membranes was best described as a partitioning of the pheromones into the lipid phase. The partitioning was enhanced by the negative surface potential of the membranes and was well described by the Gouy-Chapman theory of diffuse double layers. From the analysis of the binding of the pheromones to synthetic vesicles of known surface potential, effective charges and intrinsic partition coefficients were obtained for the pheromones. The information was used in subsequent experiments with yeast membranes to determine the intrinsic partition coefficients of the a-factor analogues and the charge density of the yeast membranes. Derivatives of a-factor with different alkyl chains in place of the normal C-terminal farnesyl displayed biological activity that paralleled the degree of partitioning of the pheromones into vesicles. Demethylation of the C-terminus decreased the partition coefficient by 6-fold and decreased the biological activity of the pheromone by greater than 2500-fold. The results show that a-factor can effectively partition into membrane bilayers and that the partitioning is probably involved in the subsequent recognition of the pheromone by the a-factor receptor.

Synthesis and biological activity of fluorescent yeast pheromones.

The mating pheromones of Saccharomyces cerevisiae and derivatives of these pheromones have been synthesized and tested for biological activity in a solution-phase assay. The effects of native alpha-factor and a-factor on the growth of target cells in these assays were identical. A derivative of alpha-factor in which the amino terminus was modified with the fluorescent probe, 6-amino-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)hexanoyl, was only slightly less active than the unmodified pheromone. Derivatives of a-factor that contain various alkyl groups in place of the farnesyl moiety of the native pheromone were also synthesized and tested for biological activity. A derivative in which the farnesyl moiety is substituted with an unbranched decyl group exhibited activity identical to that of the natural pheromone, whereas a derivative that contains an unbranched pentyl group exhibited significantly lower biological activity than native a-factor. The derivatives of a-factor have in addition been modified to incorporate 6-amino-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) at the terminus of the alkyl chains. A derivative with the probe attached to a decyl chain displayed activity similar to that of the native pheromone, whereas the same modification on a pentyl chain produced a derivative with very low activity. The fluorescence spectra of the modified alpha-factor and a-factors were measured in methanol, aqueous solution, and aqueous solution containing phospholipid vesicles. The fluorescence of the probes depends on the environment of the pheromones and can be used to monitor the association of the pheromones with the lipid bilayer.

Binding of mitochondrial presequences to yeast cytosolic heat shock protein 70 depends on the amphiphilicity of the presequence.

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.

Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanism.

The pyridoxal phosphate dependent Salmonella typhimurium dadB alanine racemase was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and O-acetyl-D-serine. Enzyme inactivation with each isomer of beta-chloro[14C]alanine followed by NaBH4 reduction and trypsin digestion afforded a single radiolabeled peptide. In the same manner, NaB3H4-reduced native enzyme gave a single labeled peptide after trypsin digestion. Purification and sequencing of these three radioactive peptides revealed them to be a common, unique hexadecapeptide which contained labeled lysine at position 6 in each case. Enzyme which had been inactivated, but not reductively stabilized with NaBH4, released a labile pyridoxal phosphate-inactivator adduct on denaturation. The structure of this adduct suggests that the enzyme was inactivated by trapping the coenzyme in a ternary adduct with inactivator and the active site lysine. Under denaturing conditions, facile alpha,beta-elimination occurred, releasing the aldol adduct of pyruvate and pyridoxal phosphate. Reduction of the ternary enzyme adduct blocked this elimination pathway. The overall mechanism of racemase inactivation is discussed in light of these results.

Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies.

Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.

A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers.

Subunit IV of yeast cytochrome oxidase is made in the cytoplasm with a transient pre-sequence of 25 amino acids which is removed upon import of the protein into mitochondria. To study the function of this cleavable pre-sequence in mitochondrial protein import, three peptides representing 15, 25 or 33 amino-terminal residues of the subunit IV precursor were chemically synthesized. All three peptides were freely soluble in aqueous buffers, yet inserted spontaneously from an aqueous subphase into phospholipid monolayers up to an extrapolated limiting monolayer pressure of 40-50 mN/m. The two longer peptides also caused disruption of unilamellar liposomes. This effect was increased by a diffusion potential, negative inside the liposomes, and decreased by a diffusion potential of opposite polarity. The peptides, particularly the two longer ones, also uncoupled respiratory control of isolated yeast mitochondria. The 25-residue peptide had little secondary structure in aqueous buffer but became partly alpha-helical in the presence of detergent micelles. Based on the amino acid sequence of the peptides, a helical structure would have a highly asymmetric distribution of charged and apolar residues and would be surface active. Amphiphilic helicity appears to be a general feature of mitochondrial pre-sequences. We suggest that this feature plays a crucial role in transporting proteins into mitochondria.

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.