Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization |
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Authors: | JC Sharpe E London |
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Institution: | (1) Department of Biochemistry and Cell Biology, Department of Chemistry, and the Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA, US |
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Abstract: | Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role
in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin
pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement
of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence.
It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends
on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released
from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration,
a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol
or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol.
The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior
may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by
membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of
rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry,
and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion
of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed.
Received: 10 March 1999/Revised: 22 June 1999 |
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Keywords: | : Fluorescence quenching — Model membranes — Membrane translocation |
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