Li Ling, Wen Maorong, Run Changqing, Wu Bin, OuYang Bo
State Key Laboratory of Molecular Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Membranes (Basel). 2022 Sep 22;12(10):916. doi: 10.3390/membranes12100916.
Mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytoplasm into the mitochondrial matrix to participate in the tricarboxylic acid (TCA) cycle, which further generates the energy for the physiological activities of cells. Two interacting subunits, MPC1 and MPC2 or MPC3, form a heterodimer to conduct transport function. However, the structural basis of how the MPC complex transports pyruvate is still lacking. Here, we described the detailed expression and purification procedures to obtain large amounts of yeast MPC1 and MPC2 for structural characterization. The purified yeast MPC1 and MPC2 were reconstituted in dodecylphosphocholine (DPC) micelles and examined using nuclear magnetic resonance (NMR) spectroscopy, showing that both subunits contain three α-helical transmembrane regions with substantial differences from what was predicted by AlphaFold2. Furthermore, the new protocol producing the recombinant MPC2 using modified maltose-binding protein (MBP) with cyanogen bromide (CNBr) cleavage introduced general way to obtain small membrane proteins. These findings provide a preliminary understanding for the structure of the MPC complex and useful guidance for further studies.
线粒体丙酮酸载体（MPC）将丙酮酸从细胞质转运到线粒体基质中，参与三羧酸（TCA）循环，进而为细胞的生理活动产生能量。两个相互作用的亚基，MPC1和MPC2或MPC3，形成异二聚体以执行转运功能。然而，MPC复合物如何转运丙酮酸的结构基础仍然缺乏。在这里，我们描述了详细的表达和纯化程序，以获得大量用于结构表征的酵母MPC1和MPC2。纯化的酵母MPC1和MPC2在十二烷基磷酸胆碱（DPC）胶束中重构，并使用核磁共振（NMR）光谱进行检测，结果表明这两个亚基都包含三个α-螺旋跨膜区域，与AlphaFold2预测的结果有很大差异。此外，使用经溴化氰（CNBr）切割的修饰麦芽糖结合蛋白（MBP）生产重组MPC2的新方案引入了获得小膜蛋白的通用方法。这些发现为MPC复合物的结构提供了初步认识，并为进一步研究提供了有用的指导。
