title: Contribution of each membrane binding domain of the CTP:phosphocholine cytidylyltransferase-alpha dimer to its activation, membrane binding, and membrane cross-bridging.
abstract: 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 full-length 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.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A-1S6, Canada.
authors.forename: Svetla
authors.initials: S
authors.lastname: Taneva
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Dennis
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Ding
authors.forename: Jillian L
authors.initials: JL
authors.lastname: Smith
authors.forename: Rosemary B
authors.initials: RB
authors.lastname: Cornell
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000737:chemistry*; D019281:Dimerization; D005976:Glutaral / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D016296:Mutagenesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D003432:Cross-Linking Reagents; D006639:Histidine; D003570:Cytidine Triphosphate; D019916:Choline-Phosphate Cytidylyltransferase; D005976:Glutaral