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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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PI-PLC inhibits the binding of proaerolysin to BHK  cells. 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. The cells  were then incubated in presence or absence of 2 nM proaerolysin  for 1 h at 4°C, thoroughly washed, and homogenized. (a) The  PNSs were probed for the presence of proaerolysin by Western  blotting. (b) The PNSs were analyzed by proaerolysin overlay for  the presence of proaerolysin binding proteins. Lane 1, control  cells; lanes 2, proaerolysin-treated cells; lane 3, PI-PLC and proaerolysin-treated cells. Arrowheads indicate proaerolysin and aerolysin (that were bound to the plasma membrane) migrating at their  expected molecular weights. Arrows indicate proaerolysin that  bound to specific proteins on the nitrocellulose membrane.
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Figure 4: PI-PLC inhibits the binding of proaerolysin to BHK cells. 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. The cells were then incubated in presence or absence of 2 nM proaerolysin for 1 h at 4°C, thoroughly washed, and homogenized. (a) The PNSs were probed for the presence of proaerolysin by Western blotting. (b) The PNSs were analyzed by proaerolysin overlay for the presence of proaerolysin binding proteins. Lane 1, control cells; lanes 2, proaerolysin-treated cells; lane 3, PI-PLC and proaerolysin-treated cells. Arrowheads indicate proaerolysin and aerolysin (that were bound to the plasma membrane) migrating at their expected molecular weights. Arrows indicate proaerolysin that bound to specific proteins on the nitrocellulose membrane.

Mentions: To identify proaerolysin-binding proteins, we have used a proaerolysin overlay assay: postnuclear supernatant (PNS) proteins were separated by SDS-PAGE, blotted onto a nitrocellulose membrane that was then incubated with proaerolysin. As shown in Fig. 2, proaerolysin bound predominantly to a protein with an apparent molecular weight of 80 kD, as well as to some lower molecular weight bands (26 and 28 kD), perhaps corresponding to degradation products. Indeed the intensity of the lower molecular weight bands varied from experiments to experiment (also see Fig. 4 b). As observed for binding of the toxin to living cells (see above), binding of 125I-proaerolysin to the proteins on the nitrocellulose membrane could be competed with an excess of unlabeled toxin. The 80-kD protein detected by this assay exhibited the biochemical properties of a membrane protein since it remained bound to a membrane fraction after higher or lower pH washes, or treatment with high salt concentrations (not shown).


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)

PI-PLC inhibits the binding of proaerolysin to BHK  cells. 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. The cells  were then incubated in presence or absence of 2 nM proaerolysin  for 1 h at 4°C, thoroughly washed, and homogenized. (a) The  PNSs were probed for the presence of proaerolysin by Western  blotting. (b) The PNSs were analyzed by proaerolysin overlay for  the presence of proaerolysin binding proteins. Lane 1, control  cells; lanes 2, proaerolysin-treated cells; lane 3, PI-PLC and proaerolysin-treated cells. Arrowheads indicate proaerolysin and aerolysin (that were bound to the plasma membrane) migrating at their  expected molecular weights. Arrows indicate proaerolysin that  bound to specific proteins on the nitrocellulose membrane.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: PI-PLC inhibits the binding of proaerolysin to BHK cells. 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. The cells were then incubated in presence or absence of 2 nM proaerolysin for 1 h at 4°C, thoroughly washed, and homogenized. (a) The PNSs were probed for the presence of proaerolysin by Western blotting. (b) The PNSs were analyzed by proaerolysin overlay for the presence of proaerolysin binding proteins. Lane 1, control cells; lanes 2, proaerolysin-treated cells; lane 3, PI-PLC and proaerolysin-treated cells. Arrowheads indicate proaerolysin and aerolysin (that were bound to the plasma membrane) migrating at their expected molecular weights. Arrows indicate proaerolysin that bound to specific proteins on the nitrocellulose membrane.
Mentions: To identify proaerolysin-binding proteins, we have used a proaerolysin overlay assay: postnuclear supernatant (PNS) proteins were separated by SDS-PAGE, blotted onto a nitrocellulose membrane that was then incubated with proaerolysin. As shown in Fig. 2, proaerolysin bound predominantly to a protein with an apparent molecular weight of 80 kD, as well as to some lower molecular weight bands (26 and 28 kD), perhaps corresponding to degradation products. Indeed the intensity of the lower molecular weight bands varied from experiments to experiment (also see Fig. 4 b). As observed for binding of the toxin to living cells (see above), binding of 125I-proaerolysin to the proteins on the nitrocellulose membrane could be competed with an excess of unlabeled toxin. The 80-kD protein detected by this assay exhibited the biochemical properties of a membrane protein since it remained bound to a membrane fraction after higher or lower pH washes, or treatment with high salt concentrations (not shown).

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