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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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The amount of toxin associated with detergent resistant membranes increases with time. BHK monolayers were incubated with 125I-proaerolysin (0.4 nM) for 1 h at 4°C, then extensively washed and further incubated for different times at 37°C. a, The amount of radioactivity associated with the cells and released into the medium was determined and expressed as a percent of the total. b, Cells were solubilized in Triton X-100 and submitted to a high-speed centrifugation. The total radioactivity in the detergent insoluble pellets and the detergent soluble supernatants were determined and expressed as a percent of the total. Error bars represent the mean of four experiments.
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Figure 3: The amount of toxin associated with detergent resistant membranes increases with time. BHK monolayers were incubated with 125I-proaerolysin (0.4 nM) for 1 h at 4°C, then extensively washed and further incubated for different times at 37°C. a, The amount of radioactivity associated with the cells and released into the medium was determined and expressed as a percent of the total. b, Cells were solubilized in Triton X-100 and submitted to a high-speed centrifugation. The total radioactivity in the detergent insoluble pellets and the detergent soluble supernatants were determined and expressed as a percent of the total. Error bars represent the mean of four experiments.

Mentions: We next quantified the fraction of the total toxin that was associated with detergent insoluble domains as a function of the incubation time at 37°C. As previously shown (Abrami et al. 1998a), 45% of the toxin initially bound to the cells at 4°C was released into the medium upon incubation at 37°C, the remaining 65%, however, was stably bound to the cells (Fig. 3 a). We then investigated whether the distribution of the cell-bound toxin would change with time. In these experiments, toxin-treated cells were incubated for different times at 37°C, then solubilized in cold Triton X-100 and submitted to a high-speed centrifugation. The amounts of toxin in the detergent insoluble pellet and in the solubilized fraction were determined. As can be seen in Fig. 3 b, the amount of toxin associated with detergent-resistant membranes increased with time, consistent with the notion that the toxin was being recruited into these microdomains, presumably because of oligomerization.


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

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

The amount of toxin associated with detergent resistant membranes increases with time. BHK monolayers were incubated with 125I-proaerolysin (0.4 nM) for 1 h at 4°C, then extensively washed and further incubated for different times at 37°C. a, The amount of radioactivity associated with the cells and released into the medium was determined and expressed as a percent of the total. b, Cells were solubilized in Triton X-100 and submitted to a high-speed centrifugation. The total radioactivity in the detergent insoluble pellets and the detergent soluble supernatants were determined and expressed as a percent of the total. Error bars represent the mean of four experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2164982&req=5

Figure 3: The amount of toxin associated with detergent resistant membranes increases with time. BHK monolayers were incubated with 125I-proaerolysin (0.4 nM) for 1 h at 4°C, then extensively washed and further incubated for different times at 37°C. a, The amount of radioactivity associated with the cells and released into the medium was determined and expressed as a percent of the total. b, Cells were solubilized in Triton X-100 and submitted to a high-speed centrifugation. The total radioactivity in the detergent insoluble pellets and the detergent soluble supernatants were determined and expressed as a percent of the total. Error bars represent the mean of four experiments.
Mentions: We next quantified the fraction of the total toxin that was associated with detergent insoluble domains as a function of the incubation time at 37°C. As previously shown (Abrami et al. 1998a), 45% of the toxin initially bound to the cells at 4°C was released into the medium upon incubation at 37°C, the remaining 65%, however, was stably bound to the cells (Fig. 3 a). We then investigated whether the distribution of the cell-bound toxin would change with time. In these experiments, toxin-treated cells were incubated for different times at 37°C, then solubilized in cold Triton X-100 and submitted to a high-speed centrifugation. The amounts of toxin in the detergent insoluble pellet and in the solubilized fraction were determined. As can be seen in Fig. 3 b, the amount of toxin associated with detergent-resistant membranes increased with time, consistent with the notion that the toxin was being recruited into these microdomains, presumably because of oligomerization.

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