Nguyen M, Argan C, Lusty C J, Shore G C
J Biol Chem. 1986 Jan 15;261(2):800-5.
Normal and hybrid proteins were synthesized following transcription of pSP64 recombinant plasmids with SP6 polymerase and translation of resultant mRNAs in a rabbit reticulocyte lysate. The precursor to the rat liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = approximately 40,000), was efficiently imported by rat heart mitochondria in vitro and processed to mature protein. Import of the precursor to a second matrix enzyme, carbamyl-phosphate synthetase I (Mr = approximately 165,000), could not be demonstrated. However, a 33-kDa hybrid protein, bearing the precarbamyl-phosphate synthetase I signal sequence (38 amino acids) and 55 amino acids from the amino terminus of the mature enzyme, followed by the carboxyl-terminal 209 amino acids of ornithine carbamyltransferase, was imported and processed under identical conditions. The topogenic function of the preornithine carbamyltransferase signal sequence (32 amino acids) was confirmed by constructing a hybrid protein bearing the signal sequence and five amino acids of mature enzyme, followed by 250 amino acids of the cytosolic enzyme of Escherichia coli, asparagine synthetase; the hybrid was transported to the matrix compartment of heart mitochondria where processing, albeit incorrect, took place. A hybrid asparagine synthetase bearing the pre-ornithine carbamyltransferase signal sequence plus 28 amino acids of mature ornithine carbamyltransferase, however, was imported and processed with apparent fidelity.
用SP6聚合酶转录pSP64重组质粒并在兔网织红细胞裂解物中翻译所得mRNA后，合成了正常蛋白和杂合蛋白。大鼠肝线粒体基质酶鸟氨酸氨甲酰基转移酶（Mr = 约40,000）的前体在体外被大鼠心脏线粒体有效导入并加工成成熟蛋白。第二种基质酶氨甲酰磷酸合成酶I（Mr = 约165,000）的前体的导入未能得到证实。然而，一种33 kDa的杂合蛋白，带有氨甲酰磷酸合成酶I前体信号序列（38个氨基酸）和成熟酶氨基末端的55个氨基酸，随后是鸟氨酸氨甲酰基转移酶的羧基末端209个氨基酸，在相同条件下被导入并加工。通过构建一种杂合蛋白证实了鸟氨酸氨甲酰基转移酶前体信号序列（32个氨基酸）的拓扑功能，该杂合蛋白带有信号序列和成熟酶的五个氨基酸，随后是大肠杆菌胞质酶天冬酰胺合成酶的250个氨基酸；该杂合体被转运到心脏线粒体的基质区室，在那里发生了加工，尽管加工不正确。然而，一种带有鸟氨酸氨甲酰基转移酶前体信号序列加28个成熟鸟氨酸氨甲酰基转移酶氨基酸的杂合天冬酰胺合成酶被导入并以明显的保真度进行了加工。
