MITOCHONDRIAL PRESEQUENCE INSERTS DIFFERENTLY INTO MEMBRANES CONTAINING CARDIOLIPIN AND PHOSPHATIDYLGLYCEROL

The interaction of the 25-residue presequence of yeast cytochrome oxidase subunit IV with lipid bilayers composed of phosphatidylglycerol, cardiolipin, or their (1:4) mixtures with phosphatidylcholine has been studied by spin-label ESR spectroscopy. Binding of the presequence progressively broadens the gel-to-fluid phase transition of dimyristoylphosphatidylglycerol bilayers, leading to abolition of the transition at a peptide/lipid ratio of greater than or equal to 1:5 mol/mol. The mobility of phosphatidylglycerol spin-labeled at the fi-position of the sn-2 chain is decreased in both gel and fluid phases on binding the presequence, with a progressively increasing ESR spectral anisotropy in the fluid phase. The ESR spectra of phosphatidylglycerol spin-labeled at the 14-position of the sn-2 chain contain a second motionally restricted component, in addition to the fluid bilayer spectral component, that arises from direct interaction of the bound presequence with the lipid chains. The proportion of this motionally restricted component is greater for dioleoylphosphatidylglycerol bilayers (corresponding to 2-3 lipids per peptide) than for cardiolipin bilayers (1-2 lipids/peptide), and this component is present also in the mixed bilayers containing 80% phosphatidylcholine. The ESR spectra of the presequence spin-labeled with a maleimide derivative at cysteine-19 evidence high mobility in solution and a very strong reduction in mobility on binding to bilayers containing negatively charged lipids. At low peptide to lipid ratios, the ESR spectra of the spin-labeled presequence sense the phase transition of dimyristoylphosphatidylglycerol. The membrane location of the spin label on the bound presequence was determined from the paramagnetic relaxation enhancements induced by lipid-permeant molecular oxygen and lipid-impermeant chromium oxalate by using calibrations established with spin-labeled lipids in the presence of presequence alkylated with N-ethylmaleimide. The label on cysteine-19 is positioned between the 5- and 14-positions of the lipid chain when bound to dioleoylphosphatidylglycerol and in the region of the 14-position for (1:4) admixture with phosphatidylcholine. When bound to cardiolipin bilayers, the location is shifted toward the membrane surface: to the headgroup region for cardiolipin alone and to the 5-position of the chain for (1:4) admixture with phosphatidylcholine. Possible functional consequences of the different localization of the presequence in phosphatidylglycerol and cardiolipin membranes are discussed.