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J. Biol. Chem., Vol. 283, Issue 15, 9562-9570, April 11, 2008

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Substrate Recognition and Binding by RseP, an Escherichia coli Intramembrane Protease^*

From the Institute for Virus Research, Kyoto University, Kawara-chu, Shogoin, Kyoto 606-8507, Japan

Escherichia coli RseP belongs to the S2P family of intramembrane cleaving proteases. RseP catalyzes proteolytic cleavage of the membrane-bound anti-^E protein RseA as an essential step in transmembrane signal transduction in the ^E extracytoplasmic stress response pathway. RseP cleaves transmembrane segments of membrane proteins, but the molecular mechanisms of its substrate recognition and proteolytic action remain largely unknown. Here we analyzed interaction between RseP and substrate membrane proteins. Co-immunoprecipitation assays showed that helix-destabilizing residues in a substrate transmembrane segment, which were previously shown to be required for efficient proteolysis of the substrate by RseP, stabilize the substrate-RseP interaction. Substitutions of certain amino acid residues, including those evolutionarily conserved, in the third transmembrane region (TM3) of RseP weakened the RseP-substrate interaction. Specific combinations of Cys substitutions in RseP TM3 and in the RseA transmembrane segment led to the formation of disulfide bonds upon oxidation, suggesting that TM3 of RseP directly binds the substrate. These results provide insights into the mechanism of membrane protein proteolysis by RseP.

Received for publication, December 7, 2007 , and in revised form, January 18, 2008.

^* This work was supported by grants from Japan Society for the Promotion of Science (to Y. A.), from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. A. and K. I.) and from CREST, Japan Science and Technology Agency (to K. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 81-75-751-4040; Fax: 81-75-771-5699; E-mail: yakiyama{at}virus.kyoto-u.ac.jp.

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