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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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Ultrastructural analysis of aerolysin-treated BHK  cells. Cells were incubated with 0.38 nM proaerolysin for 1 h at  37°C, fixed, and then processed for embedding in Epon and sectioning. A and B are low magnification overviews showing the  typical morphology of the cells. Large electron lucent vacuoles  are the striking characteristic of the treated cells. In many sections the vacuoles are clearly in continuity with the nuclear envelope (A, arrows; also see E). Together with the presence of membrane-associated ribosomes this identifies the vacuoles as part of  the ER. Despite the drastic effects on the ER morphology the  general ultrastructure of the cells is well preserved. Note that the  cytoplasm is dense, mitochondria (m; e.g., D) are not swollen,  and endosomes (e) show normal morphology (C–E). Golgi complexes are slightly swollen but still recognizable (g; C and D). n,  nucleus. Bars: (A and B) 0.5 μm; (C–E): 0.25 μm.
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Figure 11: Ultrastructural analysis of aerolysin-treated BHK cells. Cells were incubated with 0.38 nM proaerolysin for 1 h at 37°C, fixed, and then processed for embedding in Epon and sectioning. A and B are low magnification overviews showing the typical morphology of the cells. Large electron lucent vacuoles are the striking characteristic of the treated cells. In many sections the vacuoles are clearly in continuity with the nuclear envelope (A, arrows; also see E). Together with the presence of membrane-associated ribosomes this identifies the vacuoles as part of the ER. Despite the drastic effects on the ER morphology the general ultrastructure of the cells is well preserved. Note that the cytoplasm is dense, mitochondria (m; e.g., D) are not swollen, and endosomes (e) show normal morphology (C–E). Golgi complexes are slightly swollen but still recognizable (g; C and D). n, nucleus. Bars: (A and B) 0.5 μm; (C–E): 0.25 μm.

Mentions: To confirm the above morphological observations, we analyzed proaerolysin-treated BHK cells by electron microscopy. As shown in Fig. 11, the cytoplasm contained large electron lucent vacuoles. These vacuoles were already apparent in some cells after 30 min, but were obvious in all cells after 60 min (Fig. 11). In many sections the vacuoles were clearly continuous with the nuclear envelope (Fig. 11, A and E) and had ribosomes on the cytoplasmic surface, confirming that the vacuoles are derived from the rough ER. There was clearly little effect of proaerolysin on the plasma membrane permeability to protein; the cytosol of the treated cells showed the same electron density as control cells. Effects of the toxin were highly selective since the cellular ultrastructures, in particular the Golgi, endosomes, and mitochondria, were well preserved (Fig. 11, A–E).


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)

Ultrastructural analysis of aerolysin-treated BHK  cells. Cells were incubated with 0.38 nM proaerolysin for 1 h at  37°C, fixed, and then processed for embedding in Epon and sectioning. A and B are low magnification overviews showing the  typical morphology of the cells. Large electron lucent vacuoles  are the striking characteristic of the treated cells. In many sections the vacuoles are clearly in continuity with the nuclear envelope (A, arrows; also see E). Together with the presence of membrane-associated ribosomes this identifies the vacuoles as part of  the ER. Despite the drastic effects on the ER morphology the  general ultrastructure of the cells is well preserved. Note that the  cytoplasm is dense, mitochondria (m; e.g., D) are not swollen,  and endosomes (e) show normal morphology (C–E). Golgi complexes are slightly swollen but still recognizable (g; C and D). n,  nucleus. Bars: (A and B) 0.5 μm; (C–E): 0.25 μm.
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Figure 11: Ultrastructural analysis of aerolysin-treated BHK cells. Cells were incubated with 0.38 nM proaerolysin for 1 h at 37°C, fixed, and then processed for embedding in Epon and sectioning. A and B are low magnification overviews showing the typical morphology of the cells. Large electron lucent vacuoles are the striking characteristic of the treated cells. In many sections the vacuoles are clearly in continuity with the nuclear envelope (A, arrows; also see E). Together with the presence of membrane-associated ribosomes this identifies the vacuoles as part of the ER. Despite the drastic effects on the ER morphology the general ultrastructure of the cells is well preserved. Note that the cytoplasm is dense, mitochondria (m; e.g., D) are not swollen, and endosomes (e) show normal morphology (C–E). Golgi complexes are slightly swollen but still recognizable (g; C and D). n, nucleus. Bars: (A and B) 0.5 μm; (C–E): 0.25 μm.
Mentions: To confirm the above morphological observations, we analyzed proaerolysin-treated BHK cells by electron microscopy. As shown in Fig. 11, the cytoplasm contained large electron lucent vacuoles. These vacuoles were already apparent in some cells after 30 min, but were obvious in all cells after 60 min (Fig. 11). In many sections the vacuoles were clearly continuous with the nuclear envelope (Fig. 11, A and E) and had ribosomes on the cytoplasmic surface, confirming that the vacuoles are derived from the rough ER. There was clearly little effect of proaerolysin on the plasma membrane permeability to protein; the cytosol of the treated cells showed the same electron density as control cells. Effects of the toxin were highly selective since the cellular ultrastructures, in particular the Golgi, endosomes, and mitochondria, were well preserved (Fig. 11, A–E).

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