Characteristics and novel functions of SLC44A transporters

磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)是参与细胞分裂、存活和信号转导等基本过程的膜磷脂。PC和PE是通过CDP-胆碱和CDP-乙醇胺(Kennedy)途径从头合成的，在该途径中，胞外底物胆碱和乙醇胺被输送到细胞内，磷酸化，并与二酰甘油偶联形成最终的磷脂产品。虽然已经建立了多种胆碱转运系统，但乙醇胺的转运特性很差，而且还没有单一的蛋白质指定乙醇胺的转运功能。 我们研究了溶质载体44A(SLC44A)家族的转运蛋白，称为胆碱转运蛋白样蛋白-1和-2(CTL1和CTL2)。CTL1/SLC44A1是一种胆碱/质子逆向转运蛋白，为CDP-胆碱途径提供胆碱，并将胆碱氧化为甜菜碱。有迹象表明，CTL2/SLC44A2也是细胞膜和线粒体上的胆碱转运蛋白，但其功能尚未确定。我们建议 I.CTL1和CTL2是真正的质膜和线粒体乙醇胺转运体-我们将研究CTL1和CTL2在保持CTL1但缺乏CTL2的细胞中的细胞和线粒体转运，反之亦然。我们将利用CTL1/2突变体、siRNA和特异性抗体首次证明CTL1和CTL2是哺乳动物系统中的胆碱和乙醇胺转运体。 二、CTL1和CTL2调节线粒体功能-我们的初步数据表明，CTL1的幸运诱导了线粒体网络的有丝分裂和断裂。我们认为CTL1调控线粒体的融合-裂变过程。我们将研究线粒体融合和代谢的机制(电子传递复合体、ATP合成、ROS产生、膜电位等)。在CTL1和CTL2缺陷和过度表达的细胞中。最初的发现将在从CTL1和/或CTL2基因敲除小鼠中分离出来的线粒体和原代细胞中得到证实。我们希望第一次证明CTL1和CTL2是线粒体周转和线粒体能量平衡的关键调节者。 三、CTL1和CTL2结合磷脂合成与全身能量代谢--CTL1和CTL2基因敲除小鼠被培育出来，并由国际小鼠表型联盟鉴定。体重减轻和肌肉/脂肪比增加是CTL1和CTL2基因敲除小鼠的共同特征。我们将详细描述CTL1小鼠的特征，并将其与更知名的CTL2小鼠进行比较，包括调节肯尼迪途径、骨骼肌、肝脏和脂肪组织中的脂肪合成和能量代谢，应用在以前对Pcyt2基因敲除小鼠的研究中广泛使用的方案(Pcyt2是CDP-乙醇胺肯尼迪途径中的主要调节酶)。

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are membrane phospholipids involved in the fundamental processes of cell division, survival and signaling. PC and PE are synthesized de novo by the CDP-choline and CDP-ethanolamine (Kennedy) pathway, in which the extracellular substrates choline and ethanolamine are transported into the cell, phosphorylated, and coupled with diacylglycerol to form the final phospholipid product. While multiple transport systems have been established for choline, ethanolamine transport is poorly characterized, and there is no single protein assigned a transport function for ethanolamine. We investigate transporters from the family of Solute Carriers 44A (SLC44A) known as Choline Transporter-Like proteins-1 and -2 (CTL1 and CTL2). CTL1/SLC44A1 is a choline/proton antiporter and delivers choline for the CDP-choline pathway and choline oxidation to betaine. There are indications that CTL2/SLC44A2 is also a choline transporter at the plasma membrane and mitochondria, but its function is not firmly established. We propose that i. CTL1 and CTL2 are authentic plasma membrane and mitochondria ethanolamine transporters -We will characterize cellular and mitochondrial transport of CTL1 and CTl2 in cells that maintain CTL1 but lack CTL2 and vice versa. We will employ CTL1/2 mutants, siRNA and specific antibodies to demonstrate for the first time that CTL1 and CTL2 are choline and also ethanolamine transporters in mammalian systems. ii. CTL1 and CTL2 regulate mitochondrial function-Our preliminary data suggest that the luck of CTL1 induces mitophagy and fragmentation of mitochondrial networks. We propose that CTL1 regulate mitochondrial fusion-fission processes. We will investigate mechanisms of mitochondrial fusion and metabolism (electron transport complexes, ATP synthesis, ROS production, membrane potential, etc.) in CTL1 and CTL2 deficient and over-expressing cells. The initial findings will be corroborated in mitochondria and primary cells isolated from CTL1 and/or CTL2 knockout mice. We expect to demonstrate for the first time that CTL1 and perhaps CTL2 are critical regulators of mitochondrial turnover and mitochondrial energy homeostasis. iii. CTL1 and CTL2 couple phospholipid synthesis with the whole-body energy metabolism-CTL1 and CTL2 knockout mice were produced and characterized by the International Mouse Phenotyping Consortium. Reduced body weight and increased muscle/fat ratio are common features of CTL1 and CTL2 knockout mice. We will characterize CTL1 mice in full detail and compare to better known CTL2 mice, including the regulation of the Kennedy pathway, lipid synthesis and energy metabolism in skeletal muscle, liver and adipose tissue, applying protocols extensively used in previous studies of our Pcyt2 knockout mice (Pcyt2 is the main regulatory enzyme in the CDP-ethanolamine Kennedy pathway).