Molecular Studies of a Mitochondrial Ion Channel

蛋白质跨膜屏障的转运在许多细胞功能中都是重要的，包括信号、分泌、细胞器的生物发生、区域化和细胞程序性死亡。一个研究得很多但知之甚少的例子是前体蛋白被输入线粒体的过程。在这个细胞器的内膜和外膜中都有一组复杂的蛋白质识别、分类和选择性地将前体转移到它们的最终目的地。一个充满水的蛋白质孔被称为内膜的多电导通道(MCC)，被认为在这一过程中发挥了关键作用。该项目有三个与其主要目标有关的目标，即确定MCC在蛋白质进口中的生理作用及其作用的分子基础。认为MCC是进口孔的假设预测，干扰进口设备的其他组件将改变MCC的性质。因此，内膜异寡聚体输入装置的抗体处理和蛋白分解将用于测试电生理技术监测的对MCC活性的影响。MCC可能与其外膜对应的通道一起发挥作用，该通道被称为PSC(缩写为肽敏感通道)。如果外通道和内通道在蛋白质转运过程中相互作用，那么外膜复合体的结构变化也可能影响MCC的活性。因此，将分析外膜复合体的类似抗体和蛋白水解物处理对MCC活性的影响。从基因操纵的线粒体中对MCC活性进行生化重建将被用于鉴定MCC的蛋白质成分。然后对这些成分进行质谱分析，以确定它们的氨基酸序列。总之，这些研究将为蛋白质输入线粒体的机制提供基本的新信息。

Protein translocation across membrane barriers is important in many cellular functions including signaling, secretion, biogenesis of organelles, compartmentalization, and programmed cell death. A much studied yet poorly understood example is the process by which precursor proteins are imported into mitochondria. A complex set of proteins in both the inner and outer membranes of this organelle recognizes, sorts and selectively translocates precursors to their final destinations. A water-filled proteinaceous pore called the multiple conductance channel (MCC) of the inner membrane is postulated to play a key role in this process. This project has three objectives related to its main goal of determining the physiological role of the MCC in protein import and the molecular basis of its action. The hypothesis that the MCC is the import pore predicts that interfering with other components of the import apparatus will change the properties of the MCC. Therefore, treatment with antibodies and proteolysis of the hetero-oligomeric import apparatus of the inner membrane will be used to test for effects on MCC activity as monitored by electrophysiological techniques. MCC may function in tandem with its outer membrane counterpart, a channel called PSC (for peptide-sensitive channel). If the outer and inner channels interact during protein translocation, then structural changes made to the outer membrane complex may also affect MCC activity. Therefore, similar antibody and proteolysis treatments of the outer membrane complex will be analyzed for effects on MCC activity. Biochemical reconstitution of MCC activity from genetically manipulated mitochondria will be used to identify protein components of the MCC. These components will then be analyzed by mass spectrometry to determine their amino acid sequences. Together the studies will provide fundamental new information on the mechanisms of protein import into mitochondria.