Limits...
A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum.

Abrami L, Fivaz M, Glauser PE, Parton RG, van der Goot FG - J. Cell Biol. (1998)

Bottom Line: Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases.Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments.Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

View Article: PubMed Central - PubMed

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

ABSTRACT
In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

Show MeSH

Related in: MedlinePlus

Effect of proaerolysin on  the intracellular potassium concentration and the membrane potential  of BHK cells. (a) Cells were incubated with different concentrations  of proaerolysin for 1 h at 4°C, thoroughly washed and incubated with  a toxin-free medium for 15 min at  37°C. Potassium contents were determined by flame photometry. Experiments were done in triplicate,  and the standard deviation was calculated. (b) Cells were incubated  with or without 6 U/ml of PI-PLC  for 1 h at 37°C in the presence of 10  μg/ml cycloheximide, treated with or without 0.38 nM proaerolysin for 1 h at 4°C, thoroughly washed and incubated with a toxin-free  medium for 15 min at 37°C. The potassium content was then determined by flame photometry. (c) Trypsinized-BHK cells were incubated with the membrane potential–sensitive dye DiS-C3(5) as described in Materials and Methods. The arrow indicates the time at  which proaerolysin was added. Maximal depolarization was obtained at the end of each experiment by adding 1 μg/ml (final concentration) of trypsin-activated aerolysin (arrowheads). ♦, 100 ng/ml wild-type proaerolysin; □, 20 ng/ml wild-type proaerolysin; ○, 100 ng/ml  G202C-I445C proaerolysin. The slight increase in fluorescence observed in the cells treated with the G202C-I445C mutant proaerolysin  was not significant since a similar drift was observed in the absence of toxin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140172&req=5

Figure 7: Effect of proaerolysin on the intracellular potassium concentration and the membrane potential of BHK cells. (a) Cells were incubated with different concentrations of proaerolysin for 1 h at 4°C, thoroughly washed and incubated with a toxin-free medium for 15 min at 37°C. Potassium contents were determined by flame photometry. Experiments were done in triplicate, and the standard deviation was calculated. (b) Cells were incubated with or without 6 U/ml of PI-PLC for 1 h at 37°C in the presence of 10 μg/ml cycloheximide, treated with or without 0.38 nM proaerolysin for 1 h at 4°C, thoroughly washed and incubated with a toxin-free medium for 15 min at 37°C. The potassium content was then determined by flame photometry. (c) Trypsinized-BHK cells were incubated with the membrane potential–sensitive dye DiS-C3(5) as described in Materials and Methods. The arrow indicates the time at which proaerolysin was added. Maximal depolarization was obtained at the end of each experiment by adding 1 μg/ml (final concentration) of trypsin-activated aerolysin (arrowheads). ♦, 100 ng/ml wild-type proaerolysin; □, 20 ng/ml wild-type proaerolysin; ○, 100 ng/ml G202C-I445C proaerolysin. The slight increase in fluorescence observed in the cells treated with the G202C-I445C mutant proaerolysin was not significant since a similar drift was observed in the absence of toxin.

Mentions: We then investigated whether the toxin permeabilized the plasma membrane of BHK cells by measuring the intracellular potassium contents. As shown in Fig. 7 a, proaerolysin addition led to a decrease in intracellular potassium in a dose-dependent manner. No potassium efflux was observed when cells were kept at 4°C. As a control, we analyzed the effects of the G202C-I445C proaerolysin mutant, which has an engineered disulfide bridge that links the propeptide to the mature toxin (van der Goot et al., 1994). This mutant is unable to lyse erythrocytes presumably because it can no longer oligomerize. The intracellular potassium content of BHK cells was not affected by the mutant toxin, both in the proform and the mature form, even though it was able to bind to BHK cells, as shown by the fact that it could compete for binding with radiolabeled wild-type toxin (Fig. 1 a). We have also checked whether removal of GPI-anchored proteins from the cell surface would inhibit potassium efflux. PI-PLC treatment reduced the proaerolysin-induced potassium release by >75% (Fig. 7 b) again confirming that the proaerolysin receptor is GPI anchored. The remaining potassium efflux presumably reflects the fact that PI-PLC treatment was not complete.


A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum.

Abrami L, Fivaz M, Glauser PE, Parton RG, van der Goot FG - J. Cell Biol. (1998)

Effect of proaerolysin on  the intracellular potassium concentration and the membrane potential  of BHK cells. (a) Cells were incubated with different concentrations  of proaerolysin for 1 h at 4°C, thoroughly washed and incubated with  a toxin-free medium for 15 min at  37°C. Potassium contents were determined by flame photometry. Experiments were done in triplicate,  and the standard deviation was calculated. (b) Cells were incubated  with or without 6 U/ml of PI-PLC  for 1 h at 37°C in the presence of 10  μg/ml cycloheximide, treated with or without 0.38 nM proaerolysin for 1 h at 4°C, thoroughly washed and incubated with a toxin-free  medium for 15 min at 37°C. The potassium content was then determined by flame photometry. (c) Trypsinized-BHK cells were incubated with the membrane potential–sensitive dye DiS-C3(5) as described in Materials and Methods. The arrow indicates the time at  which proaerolysin was added. Maximal depolarization was obtained at the end of each experiment by adding 1 μg/ml (final concentration) of trypsin-activated aerolysin (arrowheads). ♦, 100 ng/ml wild-type proaerolysin; □, 20 ng/ml wild-type proaerolysin; ○, 100 ng/ml  G202C-I445C proaerolysin. The slight increase in fluorescence observed in the cells treated with the G202C-I445C mutant proaerolysin  was not significant since a similar drift was observed in the absence of toxin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140172&req=5

Figure 7: Effect of proaerolysin on the intracellular potassium concentration and the membrane potential of BHK cells. (a) Cells were incubated with different concentrations of proaerolysin for 1 h at 4°C, thoroughly washed and incubated with a toxin-free medium for 15 min at 37°C. Potassium contents were determined by flame photometry. Experiments were done in triplicate, and the standard deviation was calculated. (b) Cells were incubated with or without 6 U/ml of PI-PLC for 1 h at 37°C in the presence of 10 μg/ml cycloheximide, treated with or without 0.38 nM proaerolysin for 1 h at 4°C, thoroughly washed and incubated with a toxin-free medium for 15 min at 37°C. The potassium content was then determined by flame photometry. (c) Trypsinized-BHK cells were incubated with the membrane potential–sensitive dye DiS-C3(5) as described in Materials and Methods. The arrow indicates the time at which proaerolysin was added. Maximal depolarization was obtained at the end of each experiment by adding 1 μg/ml (final concentration) of trypsin-activated aerolysin (arrowheads). ♦, 100 ng/ml wild-type proaerolysin; □, 20 ng/ml wild-type proaerolysin; ○, 100 ng/ml G202C-I445C proaerolysin. The slight increase in fluorescence observed in the cells treated with the G202C-I445C mutant proaerolysin was not significant since a similar drift was observed in the absence of toxin.
Mentions: We then investigated whether the toxin permeabilized the plasma membrane of BHK cells by measuring the intracellular potassium contents. As shown in Fig. 7 a, proaerolysin addition led to a decrease in intracellular potassium in a dose-dependent manner. No potassium efflux was observed when cells were kept at 4°C. As a control, we analyzed the effects of the G202C-I445C proaerolysin mutant, which has an engineered disulfide bridge that links the propeptide to the mature toxin (van der Goot et al., 1994). This mutant is unable to lyse erythrocytes presumably because it can no longer oligomerize. The intracellular potassium content of BHK cells was not affected by the mutant toxin, both in the proform and the mature form, even though it was able to bind to BHK cells, as shown by the fact that it could compete for binding with radiolabeled wild-type toxin (Fig. 1 a). We have also checked whether removal of GPI-anchored proteins from the cell surface would inhibit potassium efflux. PI-PLC treatment reduced the proaerolysin-induced potassium release by >75% (Fig. 7 b) again confirming that the proaerolysin receptor is GPI anchored. The remaining potassium efflux presumably reflects the fact that PI-PLC treatment was not complete.

Bottom Line: Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases.Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments.Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

View Article: PubMed Central - PubMed

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

ABSTRACT
In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

Show MeSH
Related in: MedlinePlus