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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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Transport of newly synthesized VSV-G to the plasma  membrane is inhibited in proaerolysin-treated cell. Cells were infected with tsO45-VSV for 3 h 10 min at 39.5°C in the absence of  toxin. Cells were incubated an additional 20 min in IM with or  without 0.38 nM proaerolysin. Cycloheximide was added to the  cells before shifting them to 31°C for 45 min. Cells were then  fixed with paraformaldehyde and either directly decorated with  antibodies for plasma membrane staining or permeabilized with  Triton X-100 (T-X100) to visualized intracellular staining. Processing for immunofluorescence was performed as described in  Materials and Methods. Bar, 15 μm.
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Figure 15: Transport of newly synthesized VSV-G to the plasma membrane is inhibited in proaerolysin-treated cell. Cells were infected with tsO45-VSV for 3 h 10 min at 39.5°C in the absence of toxin. Cells were incubated an additional 20 min in IM with or without 0.38 nM proaerolysin. Cycloheximide was added to the cells before shifting them to 31°C for 45 min. Cells were then fixed with paraformaldehyde and either directly decorated with antibodies for plasma membrane staining or permeabilized with Triton X-100 (T-X100) to visualized intracellular staining. Processing for immunofluorescence was performed as described in Materials and Methods. Bar, 15 μm.

Mentions: Since proaerolysin induced fragmentation of the ER, affected its dynamics and led to the release of β-COP from membranes, we have investigated whether proaerolysin would affect transport to the plasma membrane of newly synthesized tsO45-G, a temperature-sensitive mutant of the transmembrane G protein of the VSV. BHK cells were infected with VSV tsO45 and incubated at the restrictive temperature (39.5°C) for 3.5 h to allow synthesis and accumulation of tsO45-G in the ER (Kreis, 1986; Kreis and Lodish, 1986). During the last 20 min of the incubation at 39.5°C, proaerolysin was added to cells. The cells were then shifted to 31°C for 45 min in the presence of cycloheximide to inhibit further protein synthesis, fixed, and then processed for immunofluorescence. As shown in Fig. 15, transport of VSV-G to the plasma membrane was dramatically inhibited. Then tsO45-G accumulated in the perinuclear region as seen after permeabilization of the fixed cells with Triton X-100 (Fig. 15). These data show that toxin action inhibits biosynthetic membrane transport of a transmembrane protein to the cell surface and suggests that the protein accumulates beyond the ER.


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)

Transport of newly synthesized VSV-G to the plasma  membrane is inhibited in proaerolysin-treated cell. Cells were infected with tsO45-VSV for 3 h 10 min at 39.5°C in the absence of  toxin. Cells were incubated an additional 20 min in IM with or  without 0.38 nM proaerolysin. Cycloheximide was added to the  cells before shifting them to 31°C for 45 min. Cells were then  fixed with paraformaldehyde and either directly decorated with  antibodies for plasma membrane staining or permeabilized with  Triton X-100 (T-X100) to visualized intracellular staining. Processing for immunofluorescence was performed as described in  Materials and Methods. Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 15: Transport of newly synthesized VSV-G to the plasma membrane is inhibited in proaerolysin-treated cell. Cells were infected with tsO45-VSV for 3 h 10 min at 39.5°C in the absence of toxin. Cells were incubated an additional 20 min in IM with or without 0.38 nM proaerolysin. Cycloheximide was added to the cells before shifting them to 31°C for 45 min. Cells were then fixed with paraformaldehyde and either directly decorated with antibodies for plasma membrane staining or permeabilized with Triton X-100 (T-X100) to visualized intracellular staining. Processing for immunofluorescence was performed as described in Materials and Methods. Bar, 15 μm.
Mentions: Since proaerolysin induced fragmentation of the ER, affected its dynamics and led to the release of β-COP from membranes, we have investigated whether proaerolysin would affect transport to the plasma membrane of newly synthesized tsO45-G, a temperature-sensitive mutant of the transmembrane G protein of the VSV. BHK cells were infected with VSV tsO45 and incubated at the restrictive temperature (39.5°C) for 3.5 h to allow synthesis and accumulation of tsO45-G in the ER (Kreis, 1986; Kreis and Lodish, 1986). During the last 20 min of the incubation at 39.5°C, proaerolysin was added to cells. The cells were then shifted to 31°C for 45 min in the presence of cycloheximide to inhibit further protein synthesis, fixed, and then processed for immunofluorescence. As shown in Fig. 15, transport of VSV-G to the plasma membrane was dramatically inhibited. Then tsO45-G accumulated in the perinuclear region as seen after permeabilization of the fixed cells with Triton X-100 (Fig. 15). These data show that toxin action inhibits biosynthetic membrane transport of a transmembrane protein to the cell surface and suggests that the protein accumulates beyond the ER.

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