Taneva Svetla, Dennis Melissa K, Ding Ziwei, Smith Jillian L, Cornell Rosemary B
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A-1S6, Canada.
J Biol Chem. 2008 Oct 17;283(42):28137-48. doi: 10.1074/jbc.M802595200. Epub 2008 Aug 11.
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.
磷酸胆碱胞苷转移酶（CCT）是磷脂酰胆碱合成中的限速酶，由一个两亲性螺旋结构域（M）介导的可逆膜相互作用调节，该结构域选择性地结合阴离子脂质。CCT是一种二聚体；因此功能单元有两个M结构域。为了探究每个M结构域的功能贡献，我们制备了一种CCT异二聚体，它由一个全长亚基与一个在M结构域之前截断且催化失活的CCT亚基配对组成。我们将这种异二聚体与全长同二聚体在阴离子囊泡激活、囊泡结合亲和力以及促进囊泡聚集方面进行了比较。令人惊讶的是，对于所有这三种功能，只有一个M结构域的二聚体的行为与有两个M结构域的二聚体相似。野生型亚基的完全激活不会因与其配对的一个M结构域缺失而受损。一个M结构域与两个M结构域的二聚体的膜结合亲和力相同，这表明二聚体的两个M结构域不会同时与单个双层膜结合。一个M结构域缺失也不妨碍囊泡交联，这表明另一个基序与M结构域协同作用以交联阴离子膜。诱变揭示带正电荷的核定位信号序列构成了膜交联的第二个基序。我们提出，CCT二聚体的两个M结构域通过交替结合机制与单个双层膜结合。系留功能涉及M结构域和核定位信号序列的协同作用，它们各自与单独的膜结合。单个M结构域的膜结合足以完全激活CCT二聚体的酶活性，同时维持调节CCT活性所需的低亲和力、可逆膜相互作用。
