Molecular Studies of a Mitochondrial Ion Channel

蛋白质跨膜屏障的易位在许多细胞功能中都很重要，包括信号传导、分泌、细胞器的生物发生、区隔化和细胞程序性死亡。一个研究得很多但了解得很少的例子是前体蛋白被输入线粒体的过程。这种细胞器的内外膜上都有一组复杂的蛋白质识别、分类并选择性地将前体转运到它们的最终目的地。一种被称为内膜多导通道（MCC）的充满水的蛋白质孔隙被认为在这一过程中起关键作用。该项目有三个目标，其主要目标是确定MCC在蛋白质进口中的生理作用及其作用的分子基础。MCC是进口孔的假设预测，干扰进口装置的其他组件将改变MCC的性质。因此，用抗体处理和内膜异聚物进口装置的蛋白水解将被用来测试对MCC活性的影响，并通过电生理技术监测。MCC可能与它的外膜对应物串联起作用，一个叫做PSC的通道（肽敏感通道）。如果外部和内部通道在蛋白质易位过程中相互作用，那么外膜复合物的结构变化也可能影响MCC的活性。因此，将分析类似的外膜复合物的抗体和蛋白水解处理对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用于鉴定MCC的蛋白质成分。这些成分将被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进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.