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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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Oligomerization of aerolysin is more efficient in vivo than in vitro. a, To follow oligomerization in vitro, proaerolysin at a concentration of 4 μM was activated with trypsin as described in Materials and Methods. The sample was then incubated at 37°C for 1 h and analyzed by SDS-PAGE, followed by Coomassie staining. The aerolysin heptamer (333 kD) is SDS resistant and can be visualized at the top of the gel. For the in vivo oligomerization experiment, 125I-proaerolysin at a concentration of 0.4 nM was activated in solution with trypsin and added to BHK cells at 4°C for 1 h. Cells were then washed and incubated for 25 min at 37°C. A PNS was prepared and analyzed by SDS-PAGE, followed by autoradiography. b, To measure potassium efflux from BHK cells at different toxin concentrations, cells were incubated with aerolysin for 1 h at 37°C and the cellular potassium contents were determined by flame photometry. Experiments were done in triplicate and the SDs were calculated.
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Figure 1: Oligomerization of aerolysin is more efficient in vivo than in vitro. a, To follow oligomerization in vitro, proaerolysin at a concentration of 4 μM was activated with trypsin as described in Materials and Methods. The sample was then incubated at 37°C for 1 h and analyzed by SDS-PAGE, followed by Coomassie staining. The aerolysin heptamer (333 kD) is SDS resistant and can be visualized at the top of the gel. For the in vivo oligomerization experiment, 125I-proaerolysin at a concentration of 0.4 nM was activated in solution with trypsin and added to BHK cells at 4°C for 1 h. Cells were then washed and incubated for 25 min at 37°C. A PNS was prepared and analyzed by SDS-PAGE, followed by autoradiography. b, To measure potassium efflux from BHK cells at different toxin concentrations, cells were incubated with aerolysin for 1 h at 37°C and the cellular potassium contents were determined by flame photometry. Experiments were done in triplicate and the SDs were calculated.

Mentions: Aerolysin is able to form heptamers in solution in the absence of membranes (van der Goot et al. 1994b; Parker et al. 1996; Rossjohn et al. 1998). However, this process is not very efficient. As can be seen in Fig. 1 a, at a toxin concentration of 4 μM, <50% of the toxin had oligomerized, even after 1 h incubation at 37°C. In marked contrast, oligomerization occurred efficiently on the surface of target cells, such as BHK cells. When BHK cells were incubated with a 104-fold lower concentration of toxin (0.4 nM), 25 min at 37°C was sufficient to observe almost complete oligomerization (Fig. 1 a, in vivo). Oligomerization and channel formation occurred at even lower doses, such as 1 pM of aerolysin, as witnessed by measuring the intracellular potassium concentration 1 h after toxin addition (Fig. 1 b). Thus, oligomerization at the cell surface can occur at bulk toxin concentrations 105–106-fold lower than those required to detect oligomerization in solution.


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

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

Oligomerization of aerolysin is more efficient in vivo than in vitro. a, To follow oligomerization in vitro, proaerolysin at a concentration of 4 μM was activated with trypsin as described in Materials and Methods. The sample was then incubated at 37°C for 1 h and analyzed by SDS-PAGE, followed by Coomassie staining. The aerolysin heptamer (333 kD) is SDS resistant and can be visualized at the top of the gel. For the in vivo oligomerization experiment, 125I-proaerolysin at a concentration of 0.4 nM was activated in solution with trypsin and added to BHK cells at 4°C for 1 h. Cells were then washed and incubated for 25 min at 37°C. A PNS was prepared and analyzed by SDS-PAGE, followed by autoradiography. b, To measure potassium efflux from BHK cells at different toxin concentrations, cells were incubated with aerolysin for 1 h at 37°C and the cellular potassium contents were determined by flame photometry. Experiments were done in triplicate and the SDs were calculated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2164982&req=5

Figure 1: Oligomerization of aerolysin is more efficient in vivo than in vitro. a, To follow oligomerization in vitro, proaerolysin at a concentration of 4 μM was activated with trypsin as described in Materials and Methods. The sample was then incubated at 37°C for 1 h and analyzed by SDS-PAGE, followed by Coomassie staining. The aerolysin heptamer (333 kD) is SDS resistant and can be visualized at the top of the gel. For the in vivo oligomerization experiment, 125I-proaerolysin at a concentration of 0.4 nM was activated in solution with trypsin and added to BHK cells at 4°C for 1 h. Cells were then washed and incubated for 25 min at 37°C. A PNS was prepared and analyzed by SDS-PAGE, followed by autoradiography. b, To measure potassium efflux from BHK cells at different toxin concentrations, cells were incubated with aerolysin for 1 h at 37°C and the cellular potassium contents were determined by flame photometry. Experiments were done in triplicate and the SDs were calculated.
Mentions: Aerolysin is able to form heptamers in solution in the absence of membranes (van der Goot et al. 1994b; Parker et al. 1996; Rossjohn et al. 1998). However, this process is not very efficient. As can be seen in Fig. 1 a, at a toxin concentration of 4 μM, <50% of the toxin had oligomerized, even after 1 h incubation at 37°C. In marked contrast, oligomerization occurred efficiently on the surface of target cells, such as BHK cells. When BHK cells were incubated with a 104-fold lower concentration of toxin (0.4 nM), 25 min at 37°C was sufficient to observe almost complete oligomerization (Fig. 1 a, in vivo). Oligomerization and channel formation occurred at even lower doses, such as 1 pM of aerolysin, as witnessed by measuring the intracellular potassium concentration 1 h after toxin addition (Fig. 1 b). Thus, oligomerization at the cell surface can occur at bulk toxin concentrations 105–106-fold lower than those required to detect oligomerization in solution.

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