Reconstitution of membrane proteins: catalysis by cholesterol of insertion of integral membrane proteins into preformed lipid bilayers

Scotto, Anthony W.; Zakim, David

doi:10.1021/bi00355a015

Cited by 34 publications

(26 citation statements)

References 36 publications

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“…This is not limited to ACT insertion. Scott and Zakim (35) observed that Ch lowered the energy barrier for insertion of integral membrane proteins into bilayers. Ch certainly favors the formation of nonlamellar structures (10,25); it also has a number of other effects on the physical properties of the bilayer.…”

Section: Discussionmentioning

confidence: 99%

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

et al. 2004

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Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium responsible for whooping cough. The 1,706-residue protein can enter eukaryotic cells, where, upon activation by endogenous calmodulin, it increases the intracellular levels of cyclic AMP, leading to severe alterations in cellular physiology, often referred to as intoxication (see reference 28 for a review). ACT belongs to the so-called RTX (repeats in toxin) family of proteins, characterized by a Ca 2ϩ -binding nonapeptide repeated in tandem several times, up to 30 to 38 repeats in the case of ACT, depending on the stringency of repeat definition. This toxin represents the most evolutionarily divergent example of the family (for reviews of RTX proteins, see references 40 and 41). Unlike most other members of the family, ACT remains associated with the bacterial surface after secretion, apparently associated with filamentous hemagglutinin (42).In common with other members of the RTX family, and apart from its unique adenylate cyclase activity, ACT has a capacity to induce cell lysis, usually demonstrated as hemolysis. ACT-induced hemolysis requires higher toxin concentrations (by more than 1 order of magnitude) and occurs more slowly than intoxication (17). Active ACT is acylated at two positions inside the chain, and the acylation pattern appears to affect hemolysis, rather than intoxication (19). Moreover, dose-response experiments suggest that intoxication can be triggered by ACT monomers, while hemolysis is a more cooperative event, mediated by at least trimers (5, 17, 32). These and other observations have led to the conclusion that hemolysis and intoxication occur through separate mechanisms (17,28,32,34).Unlike intoxication, ACT-induced cell lysis has received relatively little attention. Benz et al. (4) and Szabo et al. (39), using planar lipid bilayers, demonstrated that ACT increased membrane conductance, giving rise to small, transient, cationselective channels. These authors also found that ACT was less active in this respect than ␣-hemolysin (HlyA), another member of the RTX family, secreted by Escherichia coli (4). In general, the mechanism of HlyA-induced hemolysis has been studied in more detail (see references 16 and 40 for reviews). In particular, studies in one of our laboratories have examined the capacity of HlyA to destroy the permeability barrier of pure-lipid vesicl...

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Section: Discussionmentioning

confidence: 99%

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

et al. 2004

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“…The increased binding in the absence of DOPS could indicate that cholesterol also favours the hydrophobic interaction. It is tempting to speculate that cholesterol present in the outer mitochondrial membrane could have a stimulatory function in the binding and penetration of cytochrome c. It has been shown recently that the presence of cholesterol can catalyze the insertion of integral membrane proteins into vesicles [28]. The effect of ion concentration on the PS-apocytochrome c interaction can be in response to the screening of the surface charge, as well as in response to the effect on the protein structure or aggregation.…”

Section: Discussionmentioning

confidence: 99%

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

Pilon¹,

Jordi²,

Kruijff³

et al. 1987

Biochimica et Biophysica Acta (BBA) - Biomembranes

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(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.

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“…The membranes formed from pure PC, or PC : PG (which are cylindrically shaped phospholipids), are known to be regular liquid crystalline (L-o0bilayers, which do not exhibit the defects involved in membrane fusion (Cullis et al, 1985;Chernomordik, Melikyan & Chimadzhev, 1987). Nevertheless, the light-driven (H+)pump bacteriorhodopsin (Scotto & Zakim, 1985, 1986Rigaud et al, 1988) spontaneously inserted into phosphatidylcholine bilayer, due to the defects induced by very low amounts of detergents (Scotto & Zakim, 1985;Jain & Zakim, 1987;Rigaud et al, 1988), or fusogens (Scotto & Zakim, 1985).…”

Section: Spontaneous Insertion Of (H+)atpase Into a Preformed Bilayermentioning

confidence: 99%

“…A few membrane proteins have been successfully reconstituted into preformed liposomes by spontaneous insertion: mitochondrial ATPase (Eytan, Schatz & Racker, 1976), cyt b 5 (Enoch, Fleming & Strittmatter, 1979), cyt c oxidase (Eytan et al, 1976), UDPglucoronosyltransferase from pig liver microsomes (Scotto & Zakim, 1985), and bacteriorhodopsin (Scotto & Zakim, 1985, 1986Rigaud, Paternostre & Bluzat, 1988).…”

Section: Introductionmentioning

confidence: 99%

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer

et al. 1991

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The purified (H+)ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H(+)-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H+)ATPase molecules were closely associated with liposomes, exhibiting a high H(+)-pumping activity (150,000% quenching min-1.mg-1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H+)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H+)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles ("inside-out" orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H+)ATPase could also functionally insert into bilayer from PC:PE:PG or PC:PE:PI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H+)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H+)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation.

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Reconstitution of membrane proteins: catalysis by cholesterol of insertion of integral membrane proteins into preformed lipid bilayers

Cited by 34 publications

References 36 publications

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

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

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer

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