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Plasma membrane microdomains act as concentration platforms to facilitate intoxication by aerolysin.

Abrami L, van Der Goot FG - J. Cell Biol. (1999)

Bottom Line: Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore.We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution.Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

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β-MCD does not affect oligomerization, but dramatically accelerates protoxin processing. BHK monolayers were treated with β-MCD (10 mM in IM at 37°C for 1 h), then incubated with either 125I-proaerolysin (a) or trypsin-activated 125I-aerolysin (b; 0.4 nM) for 1 h at 4°C and subsequently incubated at 37°C for various times (indicated in minutes on the figure). PNSs were prepared and analyzed by SDS-PAGE, followed by autoradiography (20 μg of protein were loaded per lane). After β-MCD treatment, conversion of proaerolysin into aerolysin was dramatically accelerated.
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Figure 6: β-MCD does not affect oligomerization, but dramatically accelerates protoxin processing. BHK monolayers were treated with β-MCD (10 mM in IM at 37°C for 1 h), then incubated with either 125I-proaerolysin (a) or trypsin-activated 125I-aerolysin (b; 0.4 nM) for 1 h at 4°C and subsequently incubated at 37°C for various times (indicated in minutes on the figure). PNSs were prepared and analyzed by SDS-PAGE, followed by autoradiography (20 μg of protein were loaded per lane). After β-MCD treatment, conversion of proaerolysin into aerolysin was dramatically accelerated.

Mentions: Two drugs were tested: β-MCD and saponin. β-MCD is an effective extracellular cholesterol acceptor that can extract cholesterol from membranes (Kilsdonk et al. 1995; Neufeld et al. 1996). Saponin, in contrast, binds to cholesterol and sequesters it away from other interactions, but does not extract it from the membrane (Elias et al. 1978; Schroeder et al. 1998). Under the experimental conditions used, 50 ± 2% (n = 4) of total cellular cholesterol was removed by β-MCD, whereas the cholesterol content of saponin-treated cells was the same as that of control cells, as expected. After drug treatment, cells were incubated with 125I-proaerolysin for 1 h at 4°C. Neither drug prevented binding of the toxin to the cells (see Fig. 6Fig. 7Fig. 8). Toxin-treated cells were then solubilized in cold Triton X-100, submitted to a high-speed centrifugation, and the amounts of toxin in the detergent insoluble pellet and in the solubilized fraction were determined. As shown in Fig. 4 a, β-MCD led to a mild redistribution of proaerolysin to the detergent soluble fraction. However, after saponin treatment, >80% of cell bound proaerolysin was associated with the detergent soluble fraction. Similar observations were made when purifying DIGs by sucrose density centrifugation from proaerolysin-treated cells. The distribution of proaerolysin along the gradient after β-MCD treatment (results not shown) was similar to that observed for control cells (Fig. 2 a). Therefore, the toxin was still highly enriched in DIGs after β-MCD treatment, but this was no longer the case after saponin treatment (Fig. 4 b). However, saponin did not disrupt all DIGs since the caveolar marker caveolin-1 was still highly enriched in the light density detergent insoluble fractions of the gradient, as also observed for control cells (Fig. 4 c) and cells treated with β-MCD (results not shown).


Plasma membrane microdomains act as concentration platforms to facilitate intoxication by aerolysin.

Abrami L, van Der Goot FG - J. Cell Biol. (1999)

β-MCD does not affect oligomerization, but dramatically accelerates protoxin processing. BHK monolayers were treated with β-MCD (10 mM in IM at 37°C for 1 h), then incubated with either 125I-proaerolysin (a) or trypsin-activated 125I-aerolysin (b; 0.4 nM) for 1 h at 4°C and subsequently incubated at 37°C for various times (indicated in minutes on the figure). PNSs were prepared and analyzed by SDS-PAGE, followed by autoradiography (20 μg of protein were loaded per lane). After β-MCD treatment, conversion of proaerolysin into aerolysin was dramatically accelerated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164982&req=5

Figure 6: β-MCD does not affect oligomerization, but dramatically accelerates protoxin processing. BHK monolayers were treated with β-MCD (10 mM in IM at 37°C for 1 h), then incubated with either 125I-proaerolysin (a) or trypsin-activated 125I-aerolysin (b; 0.4 nM) for 1 h at 4°C and subsequently incubated at 37°C for various times (indicated in minutes on the figure). PNSs were prepared and analyzed by SDS-PAGE, followed by autoradiography (20 μg of protein were loaded per lane). After β-MCD treatment, conversion of proaerolysin into aerolysin was dramatically accelerated.
Mentions: Two drugs were tested: β-MCD and saponin. β-MCD is an effective extracellular cholesterol acceptor that can extract cholesterol from membranes (Kilsdonk et al. 1995; Neufeld et al. 1996). Saponin, in contrast, binds to cholesterol and sequesters it away from other interactions, but does not extract it from the membrane (Elias et al. 1978; Schroeder et al. 1998). Under the experimental conditions used, 50 ± 2% (n = 4) of total cellular cholesterol was removed by β-MCD, whereas the cholesterol content of saponin-treated cells was the same as that of control cells, as expected. After drug treatment, cells were incubated with 125I-proaerolysin for 1 h at 4°C. Neither drug prevented binding of the toxin to the cells (see Fig. 6Fig. 7Fig. 8). Toxin-treated cells were then solubilized in cold Triton X-100, submitted to a high-speed centrifugation, and the amounts of toxin in the detergent insoluble pellet and in the solubilized fraction were determined. As shown in Fig. 4 a, β-MCD led to a mild redistribution of proaerolysin to the detergent soluble fraction. However, after saponin treatment, >80% of cell bound proaerolysin was associated with the detergent soluble fraction. Similar observations were made when purifying DIGs by sucrose density centrifugation from proaerolysin-treated cells. The distribution of proaerolysin along the gradient after β-MCD treatment (results not shown) was similar to that observed for control cells (Fig. 2 a). Therefore, the toxin was still highly enriched in DIGs after β-MCD treatment, but this was no longer the case after saponin treatment (Fig. 4 b). However, saponin did not disrupt all DIGs since the caveolar marker caveolin-1 was still highly enriched in the light density detergent insoluble fractions of the gradient, as also observed for control cells (Fig. 4 c) and cells treated with β-MCD (results not shown).

Bottom Line: Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore.We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution.Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

Show MeSH
Related in: MedlinePlus