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Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11607-11.

Transmembrane beta-barrel of staphylococcal alpha-toxin forms in sensitive but not in resistant cells.

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1
Institute of Medical Microbiology and Hygiene, University of Mainz, D-55101 Mainz, Germany.

Abstract

Staphylococcal alpha-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118-140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118-140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126-140 entered a nonpolar environment in a striking display of an amphipathic transmembrane beta-barrel. In contrast, human granulocytes were found to bind alpha-toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.

PMID:
9326657
PMCID:
PMC23553
[Indexed for MEDLINE]
Free PMC Article
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