Hill K L, Hutchings N R, Russell D G, Donelson J E
Department of Biochemistry, University of Iowa, IA 52242, USA.
J Cell Sci. 1999 Sep;112 Pt 18:3091-101. doi: 10.1242/jcs.112.18.3091.
The flagellar pocket of African trypanosomes is a critical sorting station for protein and membrane trafficking, and is considered to be an Achilles' heel of this deadly pathogen. Although several proteins, including receptors for host-derived growth factors, are targeted specifically to the flagellar pocket, the signals responsible for this restricted subcellular localization are entirely unknown. Using T lymphocyte triggering factor-green fluorescent protein (TLTF(1)-GFP) fusion proteins, we demonstrate that an internal 144 amino acid domain of TLTF from Trypanosoma brucei is sufficient for directing GFP to the cytoplasmic side of the anterior flagellar pocket. Immuno-gold electron microscopy reveals that the TLTF-GFP fusion protein is located in an electron dense structure that immediately abuts the anterior flagellar pocket membrane. The amino acid sequence of the TLTF targeting domain does not resemble previously characterized protein trafficking signals, and random mutagenesis reveals that flagellar pocket targeting is conferred by a structural motif, rather than a short, contiguous array of amino acids. The aberrant sorting of two mutant proteins into the flagellum, and the targeting of a related human protein to the plus end of the trypanosome's cytoskeletal microtubules, lead us to suggest that flagellar pocket targeting involves interactions with the trypanosome cytoskeleton. The finding that TLTF-GFP is restricted to the anterior, cytoplasmic face of the flagellar pocket membrane, suggests that there is structural heterogeneity in the membrane of this organelle.
非洲锥虫的鞭毛袋是蛋白质和膜运输的关键分选站，被认为是这种致命病原体的致命弱点。尽管包括宿主衍生生长因子受体在内的几种蛋白质被特异性靶向鞭毛袋，但负责这种受限亚细胞定位的信号却完全未知。利用T淋巴细胞触发因子-绿色荧光蛋白（TLTF(1)-GFP）融合蛋白，我们证明来自布氏锥虫的TLTF的一个144个氨基酸的内部结构域足以将GFP导向前鞭毛袋的细胞质侧。免疫金电子显微镜显示，TLTF-GFP融合蛋白位于紧邻前鞭毛袋膜的电子致密结构中。TLTF靶向结构域的氨基酸序列与先前表征的蛋白质运输信号不同，随机诱变表明鞭毛袋靶向是由一个结构基序赋予的，而不是由短的连续氨基酸阵列赋予的。两种突变蛋白异常分选到鞭毛中，以及一种相关人类蛋白靶向锥虫细胞骨架微管的正端，这使我们认为鞭毛袋靶向涉及与锥虫细胞骨架的相互作用。TLTF-GFP局限于鞭毛袋膜的前细胞质面这一发现表明，该细胞器的膜存在结构异质性。
