Häusler T, Stierhof Y D, Wirtz E, Clayton C
Zentrum für Molekulare Biologie, Universität Heidelberg, Germany.
J Cell Biol. 1996 Feb;132(3):311-24. doi: 10.1083/jcb.132.3.311.
Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.
二氢叶酸还原酶融合蛋白已被广泛用于研究跨膜转运多肽的构象特性。我们研究了二氢叶酸还原酶融合蛋白导入布氏锥虫糖体和线粒体的过程。作为信号序列，我们将糖体磷酸甘油酸激酶的最后22个羧基末端氨基酸用于糖体，将酵母细胞色素b2或细胞色素氧化酶亚基IV的可裂解前序列用于线粒体。加入氨甲蝶呤（一种稳定二氢叶酸还原酶部分的叶酸类似物）后，靶向糖体的融合蛋白的导入未受抑制，尽管蛋白酶保护试验结果表明融合蛋白可以结合该药物。在相同条件下，氨甲蝶呤抑制了靶向线粒体的DHFR融合蛋白的导入。当同时靶向糖体和线粒体的DHFR融合蛋白表达时，氨甲蝶呤抑制其导入线粒体，而相同蛋白进入糖体的摄取不受影响或略有增加。这些发现表明，糖体具有很强的解折叠活性或异常大或灵活的转运通道。
