AhYoung Andrew P, Egea Pascal F
Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
Department of Early Discovery Biochemistry, Genentech Inc., South San Francisco, CA, USA.
Methods Mol Biol. 2019;1949:213-235. doi: 10.1007/978-1-4939-9136-5_16.
Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum-Mitochondrion Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria. Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein (TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we describe the methods for expression and purification of recombinant Mdm12, a bona fide SMP-containing protein, together with the subsequent identification of its bound phospholipids by high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as model biological membranes. These methods can be applied to the study and characterization of novel lipid-binding and lipid-transfer proteins.
内质网（ER）与线粒体之间的膜接触位点作为磷脂和钙交换的中心枢纽。酵母内质网 - 线粒体相遇结构（ERMES）复合物由五个亚基组成，这些亚基将内质网和线粒体连接在一起。三个ERMES亚基（即Mdm12、Mmm1和Mdm34）含有突触结合蛋白样线粒体脂质结合蛋白（SMP）结构域。SMP结构域属于管状脂质结合蛋白（TULIP）超家族，该超家族由普遍存在的脂质清除和转运蛋白组成。在此，我们描述了重组Mdm12（一种真正含SMP的蛋白质）的表达和纯化方法，随后通过高效薄层色谱（HPTLC）鉴定其结合的磷脂，并使用脂质置换和脂质体浮选体外测定法（以脂质体作为模型生物膜）表征其脂质交换和转运功能。这些方法可应用于新型脂质结合和脂质转运蛋白的研究与表征。
