In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.
In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.
复制标题
将萤火虫荧光素酶体内导入布氏锥虫糖体并进行 C 端靶向信号的突变分析。
DOI：
10.1091/mbc.3.7.749
复制
发表时间：
1992
影响因子：
3.3
通讯作者：
Wang,CC
中科院分区：
文献类型：
--
作者：
Sommer,JM;Cheng,QL;Keller,GA;Wang,CC
将糖酵解酶划分为专门的细胞器（糖体），使得布氏锥虫的血流形式能够仅依靠糖酵解来产生能量。这些寄生虫中糖体的生物合成已作为化疗的潜在靶标进行了深入研究。我们将最近开发的布氏锥虫稳定转化方法应用于糖体蛋白输入的体内分析。萤火虫荧光素酶是昆虫灯笼中的一种过氧化物酶体蛋白，在 T. brucei 的前环形式的稳定转化体中表达，并被发现在糖体内积聚。对位于荧光素酶C端的过氧化物酶体靶向信号丝氨酸-赖氨酸-亮氨酸(SKL)的突变分析表明，用小中性氨基酸(A、C、G、H、N、P、T)替换丝氨酸残基(Serine548)仍然导致野生型荧光素酶的50-100%的输入效率。赖氨酸 549 可以被能够形成氢键的氨基酸（H、M、N、Q、R、S）取代，而 C 端亮氨酸 550 可以被疏水性氨基酸的子集（I、M、Y）取代。因此，类似过氧化物酶体的 C 端 SKL 依赖性靶向机制可能在 T. brucei 中发挥作用，将荧光素酶导入糖体中。然而，此处鉴定的糖体靶向信号与将蛋白质引导至哺乳动物或酵母过氧化物酶体的三肽序列之间存在一些显着差异。
The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.