Komiya T, Rospert S, Schatz G, Mihara K
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
EMBO J. 1997 Jul 16;16(14):4267-75. doi: 10.1093/emboj/16.14.4267.
We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import-stimulating factor (MSF). The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor-MSF-Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor-Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl. In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor-MSF-Tom70 complex to Tom20. The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex. Formation of the Tom20-precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl. Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal. Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence.
我们在一个特定的可溶性系统中重构了前体靶向线粒体的早期步骤，该系统由酵母线粒体输入受体Tom20和Tom70的胞质结构域、牛肾上腺肾上腺皮质铁氧还蛋白前体（具有可裂解的靶向信号）以及大鼠肝脏胞质伴侣蛋白hsp70和线粒体输入刺激因子（MSF）组成。Tom70结构域仅在MSF存在时结合前体，产生前体-MSF-Tom70复合物；MSF水解ATP释放出MSF并生成前体-Tom70复合物，其形成受到过量功能性前序列肽的抑制，但不受150 mM NaCl的抑制。在Tom20结构域存在的情况下，ATP导致前体从前体-MSF-Tom70复合物转移到Tom20。单独的Tom20结构域仅在hsp70存在时结合前体；hsp70本身并未掺入形成的复合物中。Tom20-前体复合物的形成受到过量前序列肽或150 mM NaCl的抑制。对于ADP/ATP载体和孔蛋白前体也获得了类似的结果，它们都缺乏可裂解的靶向信号。因此，前体正确靶向线粒体输入受体需要胞质伴侣蛋白，而与是否存在可裂解的前序列无关。
