Interactions of mitochondrial precursor protein apocytochrome c with phosphatidylserine in model membranes. A monolayer study

Abstract

(1) The interaction of apocytochrome c with different molecular species of phosphatidyiserine was studied using monolayers at constant surface area or constant surface pressure. The protein inserted readily into dioleoylphosphatidylserine monolayers up to a limiting pressure of 50 raN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidyiserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidyiserine is indicated by the low K~ of 0.017/zM. There is little or no interaction with phosphatidylcholines. The importance of charge interactions is underlined by its ionic strength and pH dependency. (3) Experiments using ~4C-labelled apocytochrome c indicate that cholesterol can enhance the protein binding. (4) It was demonstrated that apocytochrome c monomers penetrate the monolayer whereas oligomers can be formed in an adsorbed layer and washed off without changing the surface pressure. Preincubation of apocytochrome c in 3 M guanidine, to obtain the monomeric form, was essential to measure the full effect of interracial interaction. (5) The molecular area of apocytochrome c changed from 1200-1300 .AZ/molecule in the absence of lipid to 700-900 ,A2/molecule after penetration of dioleoyiphosphatidylserine monolayers. (6) Apocytochrome c-dioleoylphosphatidyiserine interactions are only possible when the monolayer is approached from the subphase. It is concluded that the charge interactions are required for binding and penetration of the protein.

Membrane Interaction of the Glycosyltransferase MurG: a Special Role for Cardiolipin

“…One MurG molecule covers at least 20 phospholipid molecules (an estimation based on the crystal structure of MurG [14]), indicating that it is not possible that all MurG molecules are bound to these vesicles via a lipid-protein interaction. We suggest that some MurG molecules are directly bound to the lipid vesicles while others are bound in an additionally adsorbed layer via proteinprotein interactions, as was shown before for apocytochrome c (23).…”

Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxy-terminal stilbene disulfonate.

“…Cytochrome c is not only a stably folded globular protein, but is internally cross-linked: two thioether bonds originating from internal cysteine residues attach the protoheme group to the holocytochrome, thereby generating a covalent internal loop. This stable structure has frequently been invoked to explain why only the heme-free apocytochrome c interacts with lipid model systems and passes the mitochondrial outer membrane (Korb and Neupert, 1978;Rietveld and de Kruijff, 1984;Pilon et al, 1987;Dumont and Richards, 1984). While it is highly likely that the cytochrome c moiety in our cytochrome c adduct had retained the covalently bound heme group, we have not proved this directly since the small amounts of adduct available to us made such a proof difficult.…”

Domain structure and lipid interaction of recombinant yeast Tim44

“…Tim44 penetration into the monolayer was followed by measuring the surface-pressure increase of the monolayer as described (19). The experiments were performed at room temperature and, unless stated otherwise, in 20 mM Tris⅐HCl (pH 7.4) containing 100 mM NaCl.…”