Terada K, Ueda I, Ohtsuka K, Oda T, Ichiyama A, Mori M
Department of Molecular Genetics, Kumamoto University School of Medicine, Japan.
Mol Cell Biol. 1996 Nov;16(11):6103-9. doi: 10.1128/MCB.16.11.6103.
The cytosolic heat shock cognate 70-kDa protein (hsc70) is required for efficient import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria (K. Terada, K. Ohtsuka, N. Imamoto, Y. Yoneda, and M. Mori, Mol. Cell. Biol. 15:3708-3713, 1995). The requirement of hsc70 for mitochondrial import of various precursor proteins and truncated pOTCs was studied by using an in vitro translation import system in which hsc70 was completely depleted. hsc70-dependent import of pOTC was about 60% of the total import, while import of the aspartate aminotransferase precursor, the serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase was about 50, 30, and 0%, respectively. The subunit sizes of these four precursor proteins were 40 to 47 kDa. When pOTC was serially truncated from the COOH terminal, the hsc70 requirement decreased gradually and was not evident for the shortest truncated pOTCs of 90 and 72 residues. These truncated pOTCs were imported and proteolytically processed rapidly in 0.5 to 2 min at 25 degrees C, and the processed mature portions and the presequence portion were rapidly degraded. Sucrose gradient centrifugation analysis followed by import assay showed that pOTC synthesized in rabbit reticulocyte lysate forms an import-competent complex of about 11S in an hsc70-dependent manner. S values of import-competent forms of aspartate aminotransferase precursor, serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase were 9S, 9S, and 4S, respectively. Thus, the S value decreased as the hsc70 dependency decreased. Precursor proteins were coimmunoprecipitated from the reticulocyte lysate containing the newly synthesized precursor proteins with an hsc70 antibody. The amount of coimmunoprecipitated proteins was much larger in the absence of ATP than in its presence. Among the four precursor proteins, the amount of coimmunoprecipitated protein decreased as the hsc70 dependency decreased.
胞质70 kDa热休克同源蛋白（hsc70）是鸟氨酸转氨甲酰酶前体（pOTC）高效导入大鼠肝线粒体所必需的（K. 寺田、大冢健、今本直、米田洋、森正，《分子与细胞生物学》15:3708 - 3713, 1995）。通过使用hsc70完全耗尽的体外翻译导入系统，研究了hsc70对各种前体蛋白和截短的pOTC线粒体导入的需求。hsc70依赖的pOTC导入约占总导入量的60%，而天冬氨酸转氨酶前体、丝氨酸：丙酮酸转氨酶前体和3 - 氧代酰基辅酶A硫解酶的导入分别约为50%、30%和0%。这四种前体蛋白的亚基大小为40至47 kDa。当从COOH末端对pOTC进行连续截短时，hsc70需求逐渐降低，对于90和72个残基的最短截短pOTC而言不明显。这些截短的pOTC在25℃下0.5至2分钟内迅速导入并进行蛋白水解加工，加工后的成熟部分和前序列部分迅速降解。蔗糖梯度离心分析随后进行导入测定表明，兔网织红细胞裂解物中合成的pOTC以hsc70依赖的方式形成约11S的可导入复合物。天冬氨酸转氨酶前体、丝氨酸：丙酮酸转氨酶前体和3 - 氧代酰基辅酶A硫解酶的可导入形式的S值分别为9S、9S和4S。因此，随着hsc70依赖性降低，S值减小。用hsc70抗体从含有新合成前体蛋白的网织红细胞裂解物中共免疫沉淀前体蛋白。在无ATP时共免疫沉淀的蛋白量比有ATP时大得多。在这四种前体蛋白中，共免疫沉淀蛋白的量随着hsc70依赖性降低而减少。
