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J. Biol. Chem., Vol. 283, Issue 42, 28137-28148, October 17, 2008

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Contribution of Each Membrane Binding Domain of the CTP:Phosphocholine Cytidylyltransferase- Dimer to Its Activation, Membrane Binding, and Membrane Cross-bridging^*

Svetla Taneva, Melissa K. Dennis, Ziwei Ding, Jillian L. Smith¹, and Rosemary B. Cornell²

From the Departments of Molecular Biology and Biochemistry and Chemistry, Simon Fraser University, Burnaby, British Columbia V5A-1S6, Canada

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the fulllength homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.

Received for publication, April 3, 2008 , and in revised form, August 11, 2008.

^* This work was supported by Canadian Institutes of Health Research Grant 12134. 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.

¹ Present address: Genome Sciences Centre, British Columbia Cancer Agency, 570 W. 7th Ave., Vancouver, British Columbia V5Z-4S6, Canada.

² To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr., Burnaby, British Columbia V5A-1S6, Canada. Tel.: 778-782-3709; Fax: 778-782-5583; E-mail: cornell{at}sfu.ca.

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