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J. Biol. Chem., Vol. 281, Issue 10, 6448-6454, March 10, 2006

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In Vitro Reconstitution of Catabolite Repression in Escherichia coli^*

Young-Ha Park¹², Byeong R. Lee¹, Yeong-Jae Seok, Supported by the 21C Frontier Microbial Genomics and Applications Center Program Grant MG05-0202-6-0 and Korea Research Foundation Grant KRF-2004-015-C00480, Republic of Korea³, and Alan Peterkofsky^¶4

From the Department of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742, Korea, the Department of Biology, Seowon University, Chongju City 361-742, Korea, and the ^¶Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8017

A widely accepted model for catabolite repression posits that phospho-IIA^Glc of the bacterial phosphotransferase system activates adenylyl cyclase (AC) activity. For many years, attempts to observe such regulatory properties of AC in vitro have been unsuccessful. To further study the regulation, AC was produced fused to the transmembrane segments of the serine chemoreceptor Tsr. Cells harboring Tsr-AC and normal AC, expressed from the cya promoter on a low copy number vector, exhibit similar behavior with respect to elevation of cAMP levels resulting from deletion of crp, expressing the catabolite regulatory protein. Membrane-bound Tsr-AC exhibits activity comparable with the native form of AC. Tsr-AC binds IIA^Glc specifically, regardless of its phosphorylation state, but not the two general phosphotransferase system proteins, enzyme I and HPr; IIA^Glc binding is localized to the C-terminal region of AC. Binding to membranes of either dephospho- or phospho-IIA^Glc has no effect on AC activity. However, in the presence of an Escherichia coli extract, P-IIA^Glc, but not IIA^Glc, stimulates AC activity. Based on these findings of a direct interaction of IIA^Glc with AC, but activity regulation only in the presence of E. coli extract, a revised model for AC activity regulation is proposed.

Received for publication, November 28, 2005 , and in revised form, January 6, 2006.

^* This work was supported in part by the Intramural Research Program of NHLBI, National Institutes of Health. 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 Figs. 1 and 2.

¹ Both authors contributed equally to this work.

² Supported by BK21 Research Fellowships from the Korean Ministry of Education and Human Resources Development.

³To whom correspondence may be addressed. E-mail: yjseok{at}plaza.snu.ac.kr. ⁴To whom correspondence may be addressed: Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bldg. 50, Rm. 2316, MSC-8017, Bethesda, MD 20814-8017. Tel.: 301-496-2408; Fax: 301-480-0182; E-mail: peterkofsky{at}nih.gov.

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