Dual specificity of sterol-mediated glycoalkaloid induced membrane disruption.

In this study the effects of the glycoalkaloids alpha-solanine, alpha-chaconine, alpha-tomatine and the aglycone solanidine on model membranes composed of PC in the absence and presence of sterols have been analysed via permeability measurements and different biophysical methods. The main result is that glycoalkaloids are able to interact strongly with sterol containing membranes thereby causing membrane disruption in a way which is specific for the type of glycoalkaloid and sterol. For this dual specificity both the sugar moiety of the glycoalkaloid and the side-chain of the sterol on position 24 turned out to be of major importance for the membrane disrupting activity. The order of potency of the glycoalkaloids was alpha-tomatine > alpha-chaconine > alpha-solanine. The plant sterols beta-sitosterol and fucosterol showed higher affinity for glycoalkaloids as compared to cholesterol and ergosterol. The mode of action of the glycoalkaloids is proposed to consist of three main steps: (1) Insertion of the aglycone part in the bilayer. (2) Complex formation of the glycoalkaloid with the sterols present. (3) Rearrangement of the membrane caused by the formation of a network of sterol-glycoalkaloid complexes resulting in a transient disruption of the bilayer during which leakage occurs.     

A water-lipid interface induces a highly dynamic folded state in apocytochrome c and cytochrome c, which may represent a common folding intermediate.

In this study, we have used CD and NMR techniques to investigate the secondary structure of (apo-) cytochrome c both in solution and when associated with micelles. In aqueous solution, the holoprotein cytochrome c is tightly folded at secondary and tertiary levels and differs strongly from its random-coiled precursor. However, in the presence of 12-PN/12-Pglycol (9:1) micelles, we observed a remarkable resemblance between the CD spectra of these partially helical proteins. The water-lipid interface induces a secondary folding of apocytochrome c, whereas cytochrome c is suggested to partially lose its tertiary structure. The exchange of all amide protons and, using deuterium-labeled proteins, of all amide deuterons with the solvent was monitored by NMR. A rapid exchange rate was observed, indicating that these folding states are highly dynamic. Saturation-transfer NMR of micelle-associated apocytochrome c showed that the exchange takes place at the (sub-) second time scale. The holoprotein in the presence of micelles was found to have two distinct exchange rates: (1) a fast rate, comparable to that found for the micelle-associated precursor and 4.5 times slower than that of the random-coiled apocytochrome c, and (2) a slow rate which is 75 times slower than the precursor in solution. Urea denaturation studies showed the micelle-bound proteins to have a low helix stability, which explains the inability of the lipid-induced secondary structure to prevent its labile protons from rapid exchange. The uniqueness of this lipid-induced highly dynamic folding state of (apo-) cytochrome c is demonstrated by comparison with amphiphilic polypeptides like melittin, and its implications for membrane translocation and functioning are discussed.     

In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex.

We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel-filtration techniques. It is demonstrated that prePhoE rapidly adopts beta structure, folds and aggregates upon dilution to urea concentrations below 3 M. These processes are paralleled by a loss of translocation competence. Furthermore the interaction of prePhoE with SecB was investigated. SecB is shown to have a very high content of beta structure, therefore we propose that precursor recognition by SecB is mediated through beta-beta interaction. It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea. However, SecB prevents the precursor from aggregating by forming a functional and stable complex.     

Bilayer-penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria.

In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV-DHFR, which exhibit mitochondrial import pathways with different characteristics. In-vitro-synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein [Rietveld, A. & de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6707; Dumont, M. E. & Richards, F. M. (1984) J. Biol. Chem. 259, 4147-4156]. Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in-vitro-synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria. In contrast, the purified and in-vitro-synthesized precursor fusion protein pCOX IV-DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer. The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins. The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.     

Determination of the size of the packing defects in dimyristoylphosphatidylcholine bilayers,present at the phase transition temperature

Multilamellar liposomes of dimyristoylphosphatidylcholine, containing 4 mol% egg phosphatidic acid show at the phase transition temperature an increased permeability for non-electrolytes of M_r values up to 900. This indicates that the packing defects occurring at the liquid crystalline/gel state phase boundary have a similar pore diameter (15–18 A) as the packing defects present in glycophorin—dioleoylphos-phatidylcholine vesicles. This suggests that packing defects at the protein—lipid interphase are the major permeation pathway of the glycophorin—dioleoylphosphatidylcholine vesicles.     

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

(1) The interaction of apocytochrome c with different molecular species of phosphatidylserine 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 mN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidylserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidylserine is indicated by the low Kd of 0.017 microM. 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 14C-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 interfacial interaction. (5) The molecular area of apocytochrome c changed from 1200-1300 A2/molecule in the absence of lipid to 700-900 A2/molecule after penetration of dioleoylphosphatidylserine monolayers. (6) Apocytochrome c-dioleoylphosphatidylserine 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.     

The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles

Band 3 protein has been incorporated into lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine or total erythrocyte lipids by means of a Triton X-100 Bio-Beads method, with an additional sonication step prior to the removal of the detergent. This methods results, for both types of band 3 lipid vesicles, in rather homogeneous vesicles with comparable protein content and vesicle trap. Freeze-fracture electron microscopy revealed that band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have considerably more intramembrane particles as compared to the band 3-erythrocyte lipid vesicles. The dimensions of the nonspecific permeation pathways present in the band 3-lipid vesicles were measured using an influx assay procedure for nonelectrolytes of different size, in which the vesicles were sampled and subsequently freed from nonenclosed labeled permeant by means of gel-filtration. The band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have nonspecific permeation pathways (pores), with diameters of up to 60 A. In contrast, the band 3-total erythrocyte lipid vesicles are more homogeneous and show much smaller nonspecific permeation pathways, having a diameter of about 12 A. These results suggest that the nonspecific permeability of the band 3-lipid vesicles is strongly lipid-dependent. Increase in specific anion permeability expected as a consequence of the presence of band 3 in the erythrocyte lipid vesicles was found to be very limited. However, stereospecific, phloretin-inhibitable D-glucose permeability could clearly be demonstrated in these vesicles. The difference of the nonspecific permeability of the band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles and band 3-erythrocyte lipid vesicles, is discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce formation of pores.     

SecB protein stabilizes a translocation-competent state of purified prePhoE protein

Efficient translocation of pure precursor of PhoE protein (prePhoE) could be accomplished in an in vitro system consisting of only inverted Escherichia coli inner membrane vesicles, ATP, and SecA and SecB protein. In this in vitro system SecB and not trigger factor could stabilize a translocation-competent state of prePhoE. In contrast, translocation competency of proOmpA could be induced by both trigger factor and SecB protein, suggesting specificity in interactions between cytosolic factors and precursors in outer membrane protein translocation.     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.