Schleiff E, Turnbull J L
Department of Biochemistry, McGill University, Montreal, Canada.
Biochemistry. 1998 Sep 22;37(38):13043-51. doi: 10.1021/bi9807456.
Tom20 is an outer mitochondrial membrane protein that functions as a component of the import receptor complex for cytoplasmically synthesized mitochondrial precursor proteins. The human homologue, hTom20, consists of an N-terminal membrane anchor region predicted between aa5-25 and a soluble cytosolic domain from aa30 to 145. To analyze the properties of hTom20, we have expressed several truncations of the cytosolic domain as fusion proteins with glutathione S-transferase. Our studies reveal that the cytosolic region of hTom20 is a monomeric protein in solution containing two domains which are involved in different functions of the receptor. The N-terminal region is involved in membrane binding (aa30-60) and recognition of the cleavable matrix targeting signals (aa50-90). In addition, we have demonstrated that the receptor recognizes the alpha-helical state of the matrix targeting signal. The dissociation constant for this interaction in the presence of a detergent which induces this secondary structure is 0.6 microM, one-fifth the value in the absence of detergent. In aqueous solution, the region between aa30 and 60 is loosely folded and stabilized against proteolytic cleavage by interaction with detergents or a matrix targeting signal. Our work further shows that the remainder of the cytosolic domain of hTom20, aa60-145, is a compactly folded globular domain containing a region (aa90-145) that is critical for the recognition of proteins bearing internal signal sequences such as the uncoupling protein and porin.
Tom20是一种线粒体外膜蛋白，作为细胞质合成的线粒体前体蛋白导入受体复合物的一个组成部分发挥作用。人类同源物hTom20由预测位于第5至25位氨基酸之间的N端膜锚定区域和第30至145位氨基酸的可溶性胞质结构域组成。为了分析hTom20的特性，我们将胞质结构域的几个截短片段作为与谷胱甘肽S-转移酶的融合蛋白进行了表达。我们的研究表明，hTom20的胞质区域在溶液中是一种单体蛋白，包含两个参与受体不同功能的结构域。N端区域参与膜结合（第30至60位氨基酸）和可裂解基质靶向信号的识别（第50至90位氨基酸）。此外，我们已经证明该受体识别基质靶向信号的α螺旋状态。在诱导这种二级结构的去污剂存在下，这种相互作用的解离常数为0.6微摩尔，是不存在去污剂时该值的五分之一。在水溶液中，第30至60位氨基酸之间的区域折叠松散，通过与去污剂或基质靶向信号的相互作用而稳定，抵抗蛋白水解切割。我们的工作进一步表明，hTom20胞质结构域的其余部分，即第60至145位氨基酸，是一个紧密折叠的球状结构域，包含一个对识别带有内部信号序列的蛋白质（如解偶联蛋白和孔蛋白）至关重要的区域（第90至145位氨基酸）。
