Abstract
Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G. (1991) J. Biol. Chem. 266, 21693-21699). Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20. A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2. Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import. Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein. Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition. F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25%. In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25%. Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi. Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions. F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step. Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide. We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria.
Highlights
20, corresponding to thNe Hz-terminal 20 amino acids post-translationally into the appropriate suborganellar comof the F1.ATPase B-subunit precursorshow that this peptide binds avidly tophospholipid bi
Importance of amphiphilicity in the import signal experiments withvalinomycin demonstrate that when has been demonstrated by experiments in which artificial mitochondrial A$ is reduced 25% import of pFl@is mitochondrial presequences, made from serine, leucine, and inhibited only 25%
Therefoaret,least 75%of maximal arginine, were found to direct import of passenger proteins import inhibition observed in the presence of F1B132+2 andF1B21-51+3 does notresult from dissipation of A$
Summary
PM,Hoyt et al, 1991).studies with the preornithine carbamyltransferase presequence, amino acids 1-27, show that this peptide can completely block mitochondrial import at 5-10 PM,and collapse A# across the inner mitochondrial membrane unless excess reticulocyte lysate is present in the import reactions (Gillespie et al, 1985). In vitro import of "S-labeled precursor protein was carried out in 100-plreaction mixtures containing 200 mM sucrose, 10 mM HEPES, pH 7.4, 100 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 1 mM ATP, 10 mM succinate, 25 mM creatine phosphate, 2.5 mg/ml of creatine phosphokinase, 3% BSA, and mitochondria (4060 pg), thawed on ice just before use. Others have shown that import information in addition to the presequence is present within the first 34 amino acidsof preF,P (Redwell et al, 1987) andthus, least 25 min
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