Identification and characterization of protein N-myristoylation occurring on four human mitochondrial proteins, SAMM50, TOMM40, MIC19, and MIC25

Previously, we showed that SAMM50, a mitochondrial outer membrane protein, is N-myristoylated, and this lipid modification is required for the proper targeting of SAMM50 to mitochondria. In this study, we characterized protein N-myristoylation occurring on four human mitochondrial proteins, SAMM50, TOMM40, MIC19, and MIC25, three of which are components of the mitochondrial intermembrane space bridging (MIB) complex, which plays a critical role in the structure and function of mitochondria. In vitro and in vivo metabolic labeling experiments revealed that all four of these proteins were N-myristoylated. Analysis of intracellular localization of wild-type and non-myristoylated G2A mutants of these proteins by immunofluorescence microscopic analysis and subcellular fractionation analysis indicated that protein N-myristoylation plays a critical role in mitochondrial targeting and membrane binding of two MIB components, SAMM50 and MIC19, but not those of TOMM40 and MIC25. Immunoprecipitation experiments using specific antibodies revealed that MIC19, but not MIC25, was a major N-myristoylated binding partner of SAMM50. Immunoprecipitation experiments using a stable transformant of MIC19 confirmed that protein N-myristoylation of MIC19 is required for the interaction between MIC19 and SAMM50, as reported previously. Thus, protein N-myristoylation occurring on two mitochondrial MIB components, SAMM50 and MIC19, plays a critical role in the mitochondrial targeting and protein-protein interaction between these two MIB components.

此前本团队已有研究证实，线粒体外膜蛋白SAMM50发生N-肉豆蔻酰化（N-myristoylation）修饰，且该脂质修饰是SAMM50精准靶向线粒体的必要条件。本研究中，我们对4种人类线粒体蛋白的N-肉豆蔻酰化修饰进行了系统表征，这4种蛋白分别为SAMM50、TOMM40、MIC19与MIC25，其中3种属于线粒体膜间间隙桥接复合体（mitochondrial intermembrane space bridging, MIB）的组成成分，该复合体在线粒体的结构与功能调控中发挥关键作用。体外（in vitro）与体内（in vivo）代谢标记实验结果显示，上述4种蛋白均发生了N-肉豆蔻酰化修饰。通过免疫荧光显微镜分析（immunofluorescence microscopic analysis）与亚细胞分级分离分析（subcellular fractionation analysis），对上述蛋白的野生型（wild-type）及非肉豆蔻酰化G2A突变体（non-myristoylated G2A mutant）的细胞内定位进行检测后发现，蛋白质N-肉豆蔻酰化修饰对两种MIB复合体组分——SAMM50与MIC19的线粒体靶向及膜结合过程具有关键调控作用，但对TOMM40与MIC25并无此类影响。使用特异性抗体开展的免疫沉淀实验（immunoprecipitation experiment）结果显示，MIC19（而非MIC25）是SAMM50的主要N-肉豆蔻酰化结合伴侣。利用MIC19的稳定转化株（stable transformant）进行的免疫沉淀实验进一步证实，MIC19的N-肉豆蔻酰化修饰是其与SAMM50发生相互作用的必要条件，这与此前的研究报道相符。综上，两种线粒体MIB复合体组分SAMM50与MIC19上发生的蛋白质N-肉豆蔻酰化修饰，对二者的线粒体靶向及蛋白质-蛋白质相互作用（protein-protein interaction）均具有关键调控作用。