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Journal of Bacteriology, September 2002, p. 4747-4756, Vol. 184, No. 17
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.17.4747-4756.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Stochastic Assembly of Two-Component Staphylococcal {gamma}-Hemolysin into Heteroheptameric Transmembrane Pores with Alternate Subunit Arrangements in Ratios of 3:4 and 4:3

Noriko Sugawara-Tomita, Toshio Tomita, and Yoshiyuki Kamio*

Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan

Received 18 April 2002/ Accepted 7 June 2002

Self-assembling, pore-forming toxins from Staphylococcus aureus are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component {gamma}-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione S-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan. Phone: 81-22-717-8779. Fax: 81-22-717-8780. E-mail: ykamio{at}biochem.tohoku.ac.jp.

Journal of Bacteriology, September 2002, p. 4747-4756, Vol. 184, No. 17
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.17.4747-4756.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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