Gaume B, Klaus C, Ungermann C, Guiard B, Neupert W, Brunner M
Institut für Physiologische Chemie der Universität München, Goethestrasse 33, 80336 München, Germany.
EMBO J. 1998 Nov 16;17(22):6497-507. doi: 10.1093/emboj/17.22.6497.
Unfolding of preproteins and translocation across the mitochondrial membranes requires their interaction with mt-Hsp70 and Tim44 at the inner face of the inner membrane and ATP as an energy source. We measured the temperature dependence of the rates of unfolding and import into the matrix of two folded passenger domains, the tightly folded heme-binding domain (HBD) of cytochrome b2 and the loosely folded mouse dihydrofolate reductase (DHFR). Despite the stability of the HBD, its rates of thermal breathing were fast and the preprotein was imported rapidly at all temperatures. In contrast, rates of unfolding and import of DHFR were strongly temperature dependent and import was significantly slower than unfolding. In addition, import rates of DHFR were strongly dependent on the length of the presequence. We propose that the mitochondrial import motor does not exert a constant pulling force. Rather, mt-Hsp70 appears to release a translocating polypeptide chain such that the precursor can then slide back and refold on the surface of the mitochondria. Refolding competes with translocation, and passengers may undergo several rounds of unfolding and refolding prior to their import.
前体蛋白的解折叠以及跨线粒体膜的转运需要它们在内膜内表面与线粒体热休克蛋白70（mt-Hsp70）和Tim44相互作用，并以ATP作为能量来源。我们测量了两种折叠的乘客结构域（细胞色素b2紧密折叠的血红素结合结构域（HBD）和松散折叠的小鼠二氢叶酸还原酶（DHFR））的解折叠速率以及导入线粒体基质的温度依赖性。尽管HBD很稳定，但其热呼吸速率很快，并且前体蛋白在所有温度下都能快速导入。相比之下，DHFR的解折叠和导入速率强烈依赖于温度，并且导入明显比解折叠慢。此外，DHFR的导入速率强烈依赖于前导序列的长度。我们提出，线粒体导入马达不会施加恒定的拉力。相反，mt-Hsp70似乎会释放正在转运的多肽链，使得前体蛋白随后能够在线粒体表面上向后滑动并重新折叠。重新折叠与转运相互竞争，并且乘客结构域在导入之前可能会经历几轮解折叠和重新折叠。
