Law R H, Nagley P
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Biochim Biophys Acta. 1990 Aug 24;1027(2):141-8. doi: 10.1016/0005-2736(90)90077-2.
We have studied the import into isolated yeast mitochondria of three hydrophobic passenger proteins attached to the N-terminal cleavable presequence of mitochondrial ATPase subunit 9 from Neurospora crassa. One natural precursor (pN9) contained N. crassa subunit 9; two chimaeric precursors, N9L/Y8-1 and N9L/Y9-2, respectively contained yeast mitochondrial ATPase subunits 8 and 9. In the absence of urea, pN9 and N9L/Y8-1 are imported efficiently but N9L/Y9-2 is not imported. After pretreatment of precursors in 4 M urea, binding of pN9 to mitochondria is marginally affected while its import is substantially inhibited; the binding to mitochondria of chimaeric proteins, N9L/Y8-1 and N9L/Y9-2, is greatly enhanced but no import is observed. This behaviour of import precursors containing hydrophobic passenger proteins is contrasted with that of a hydrophilic chimaeric precursor pCOXIV-DHFR, whose binding and import are enhanced by pretreatment with a high concentration of urea (8 M). The import of N9L/Y8-1 is very sensitive to the presence of low concentrations of urea in the import reaction mixture, and is abolished above 0.5 M urea although precursor binding to mitochondria is increased. By contrast, neither the import nor binding of pCOXIV-DHFR is affected directly by urea up to 0.8 M. These deleterious effects of urea on import of the chimaeric precursors N9L/Y8-1 and N9L/Y9-2 are interpreted in terms of a non-productive binding of these precursors to mitochondria, brought about by exposure of their hydrophobic domains resulting from urea unfolding. The generalization that membrane translocation of mitochondrial import precursors is enhanced by their prior unfolding in urea thus does not apply in the case of these precursors containing hydrophobic passenger proteins.
我们研究了三种与来自粗糙脉孢菌线粒体ATP酶亚基9的N端可裂解前序列相连的疏水乘客蛋白导入分离的酵母线粒体的情况。一种天然前体（pN9）包含粗糙脉孢菌亚基9；两种嵌合前体N9L/Y8-1和N9L/Y9-2分别包含酵母线粒体ATP酶亚基8和9。在没有尿素的情况下，pN9和N9L/Y8-1能高效导入，但N9L/Y9-2不能导入。在前体用4M尿素预处理后，pN9与线粒体的结合仅受到轻微影响，但其导入受到显著抑制；嵌合蛋白N9L/Y8-1和N9L/Y9-2与线粒体的结合大大增强，但未观察到导入。含有疏水乘客蛋白的导入前体的这种行为与亲水嵌合前体pCOXIV-DHFR的行为形成对比，后者的结合和导入通过用高浓度尿素（8M）预处理而增强。N9L/Y8-1的导入对导入反应混合物中低浓度尿素的存在非常敏感，在尿素浓度高于0.5M时导入被消除，尽管前体与线粒体的结合增加。相比之下，高达0.8M的尿素对pCOXIV-DHFR的导入和结合均无直接影响。尿素对嵌合前体N9L/Y8-1和N9L/Y9-2导入的这些有害影响被解释为这些前体与线粒体的非生产性结合，这是由尿素展开导致其疏水结构域暴露所引起的。因此，线粒体导入前体在尿素中预先展开会增强其膜转运这一普遍规律不适用于这些含有疏水乘客蛋白的前体。
