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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.

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Binding of 125I-proaerolysin to BHK cells. (a) Inhibition of 125I-proaerolysin binding to BHK cells by unlabeled toxin.  Cells were incubated for 1 h with 4 nM of 125I-proaerolysin plus  the indicated amount of unlabeled wild-type toxin (▪). 50% inhibition occurred in the presence of 16 nM of unlabeled proaerolysin. Bars indicate the standard deviation (n = 3). Binding of  wild-type 125I-proaerolysin could also be competed with an excess  of unlabeled G202C-I445C mutant proaerolysin (□). (b) Kinetics  of specific binding of 125I-proaerolysin to BHK cells at 4°C. Cells  were incubated with 4 nM 125I-proaerolysin and washed at the indicated times. Specifically bound proaerolysin was determined by  subtracting the values obtained in the presence of 50-fold excess  of unlabeled toxin. Bars indicate the standard deviation (n = 3).  (c) Concentration dependence of proaerolysin binding to BHK  cells. Cells were incubated for 2.5 h at 4°C with 125I-proaerolysin  in the absence (○) or in the presence of 50-fold excess unlabeled  toxin (□). Specific binding (♦) was calculated by subtraction of  the unspecific bound toxin (□) from the total bound toxin (○).  The results represent the mean of two independent experiments.  Maximal errors were <16%.
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Figure 1: Binding of 125I-proaerolysin to BHK cells. (a) Inhibition of 125I-proaerolysin binding to BHK cells by unlabeled toxin. Cells were incubated for 1 h with 4 nM of 125I-proaerolysin plus the indicated amount of unlabeled wild-type toxin (▪). 50% inhibition occurred in the presence of 16 nM of unlabeled proaerolysin. Bars indicate the standard deviation (n = 3). Binding of wild-type 125I-proaerolysin could also be competed with an excess of unlabeled G202C-I445C mutant proaerolysin (□). (b) Kinetics of specific binding of 125I-proaerolysin to BHK cells at 4°C. Cells were incubated with 4 nM 125I-proaerolysin and washed at the indicated times. Specifically bound proaerolysin was determined by subtracting the values obtained in the presence of 50-fold excess of unlabeled toxin. Bars indicate the standard deviation (n = 3). (c) Concentration dependence of proaerolysin binding to BHK cells. Cells were incubated for 2.5 h at 4°C with 125I-proaerolysin in the absence (○) or in the presence of 50-fold excess unlabeled toxin (□). Specific binding (♦) was calculated by subtraction of the unspecific bound toxin (□) from the total bound toxin (○). The results represent the mean of two independent experiments. Maximal errors were <16%.

Mentions: As a first step in the characterization of the interaction of proaerolysin with mammalian cells, we have analyzed the binding of 125I-labeled proaerolysin to BHK cells. All experiments were performed at 4°C to prevent possible internalization of the toxin. A detailed analysis of competition with unlabeled toxin showed that 125I-proaerolysin could bind to the cells, and that this binding was progressively inhibited at increasing concentrations of unlabeled toxin (Fig. 1, a); thus indicating that binding was specific. That binding was of high affinity is illustrated by the fact that 16 nM of unlabeled proaerolysin could inhibit binding of 125I-proaerolysin (4 nM) by 50%. Binding could also be inhibited by an excess of the hemolytically inactive G202C-I445C proaerolysin mutant (see below; van der Goot et al., 1994). Association of 125I-proaerolysin to BHK cells reached a plateau after 2 h (Fig. 1 b). Finally, binding was saturable, a maximum being reached at a 125I-proaerolysin concentration of about 50 nM (Fig. 1 c). Binding was essentially irreversible in the time range of our experiments since <10% of 125I-proaerolysin was released after 4 h of incubation in a toxin-free medium (not shown). These experiments show that 125I-proaerolysin binds to high affinity sites on BHK cells.


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)

Binding of 125I-proaerolysin to BHK cells. (a) Inhibition of 125I-proaerolysin binding to BHK cells by unlabeled toxin.  Cells were incubated for 1 h with 4 nM of 125I-proaerolysin plus  the indicated amount of unlabeled wild-type toxin (▪). 50% inhibition occurred in the presence of 16 nM of unlabeled proaerolysin. Bars indicate the standard deviation (n = 3). Binding of  wild-type 125I-proaerolysin could also be competed with an excess  of unlabeled G202C-I445C mutant proaerolysin (□). (b) Kinetics  of specific binding of 125I-proaerolysin to BHK cells at 4°C. Cells  were incubated with 4 nM 125I-proaerolysin and washed at the indicated times. Specifically bound proaerolysin was determined by  subtracting the values obtained in the presence of 50-fold excess  of unlabeled toxin. Bars indicate the standard deviation (n = 3).  (c) Concentration dependence of proaerolysin binding to BHK  cells. Cells were incubated for 2.5 h at 4°C with 125I-proaerolysin  in the absence (○) or in the presence of 50-fold excess unlabeled  toxin (□). Specific binding (♦) was calculated by subtraction of  the unspecific bound toxin (□) from the total bound toxin (○).  The results represent the mean of two independent experiments.  Maximal errors were <16%.
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Related In: Results  -  Collection

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Figure 1: Binding of 125I-proaerolysin to BHK cells. (a) Inhibition of 125I-proaerolysin binding to BHK cells by unlabeled toxin. Cells were incubated for 1 h with 4 nM of 125I-proaerolysin plus the indicated amount of unlabeled wild-type toxin (▪). 50% inhibition occurred in the presence of 16 nM of unlabeled proaerolysin. Bars indicate the standard deviation (n = 3). Binding of wild-type 125I-proaerolysin could also be competed with an excess of unlabeled G202C-I445C mutant proaerolysin (□). (b) Kinetics of specific binding of 125I-proaerolysin to BHK cells at 4°C. Cells were incubated with 4 nM 125I-proaerolysin and washed at the indicated times. Specifically bound proaerolysin was determined by subtracting the values obtained in the presence of 50-fold excess of unlabeled toxin. Bars indicate the standard deviation (n = 3). (c) Concentration dependence of proaerolysin binding to BHK cells. Cells were incubated for 2.5 h at 4°C with 125I-proaerolysin in the absence (○) or in the presence of 50-fold excess unlabeled toxin (□). Specific binding (♦) was calculated by subtraction of the unspecific bound toxin (□) from the total bound toxin (○). The results represent the mean of two independent experiments. Maximal errors were <16%.
Mentions: As a first step in the characterization of the interaction of proaerolysin with mammalian cells, we have analyzed the binding of 125I-labeled proaerolysin to BHK cells. All experiments were performed at 4°C to prevent possible internalization of the toxin. A detailed analysis of competition with unlabeled toxin showed that 125I-proaerolysin could bind to the cells, and that this binding was progressively inhibited at increasing concentrations of unlabeled toxin (Fig. 1, a); thus indicating that binding was specific. That binding was of high affinity is illustrated by the fact that 16 nM of unlabeled proaerolysin could inhibit binding of 125I-proaerolysin (4 nM) by 50%. Binding could also be inhibited by an excess of the hemolytically inactive G202C-I445C proaerolysin mutant (see below; van der Goot et al., 1994). Association of 125I-proaerolysin to BHK cells reached a plateau after 2 h (Fig. 1 b). Finally, binding was saturable, a maximum being reached at a 125I-proaerolysin concentration of about 50 nM (Fig. 1 c). Binding was essentially irreversible in the time range of our experiments since <10% of 125I-proaerolysin was released after 4 h of incubation in a toxin-free medium (not shown). These experiments show that 125I-proaerolysin binds to high affinity sites on BHK cells.

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