Miyazawa S, Osumi T, Hashimoto T, Ohno K, Miura S, Fujiki Y
Department of Biochemistry, Shinshu University School of Medicine, Nagano, Japan.
Mol Cell Biol. 1989 Jan;9(1):83-91. doi: 10.1128/mcb.9.1.83-91.1989.
To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.
为了鉴定大鼠肝脏过氧化物酶体酰基辅酶A氧化酶（AOX）的拓扑信号，我们用该酶的突变多肽进行了体外导入实验。通过对外部添加的蛋白酶K的抗性测定，全长AOX和在N端区域截短的多肽能有效地导入过氧化物酶体。在C端区域带有内部缺失的多肽表现出低得多的导入活性。在极端C端截短或突变的多肽完全没有导入活性。当将极端C端的五个氨基酸残基连接到一些无导入活性的多肽上时，导入活性得以恢复。此外，AOX的C端199和70个氨基酸残基将与两种细菌酶的融合蛋白导向过氧化物酶体。这些结果被解释为意味着AOX的过氧化物酶体靶向信号位于C端，且极端末端的五个或更少残基在靶向中具有必需功能。C端内部区域对于有效导入也具有重要作用，可能是通过构象效应。
