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D Roise

Publications and source records attributed to D Roise.

7 recordsLinked to original sources
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1989

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)

Animals↗
PubMed1989

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.

Choline↗
PubMed1988

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.

Circular Dichroism↗
PubMed1986

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.

Amino Acid Sequence↗