Field H, Field M C
Laboratory of Cell Biology, Department of Biochemistry, Imperial College of Science, Technology, and Medicine, Exhibition Road, London SW7 2AY, United Kingdom.
J Biol Chem. 1997 Apr 18;272(16):10498-505. doi: 10.1074/jbc.272.16.10498.
The Ras superfamily of small G proteins governs unidirectional cellular processes by virtue of GTP hydrolysis and concomitant conformational changes, which are in turn regulated by a number of accessory factors. Members of the Rab subfamily are important for correct targeting and fusion of intra-organellar vesicles loaded with trafficking proteins and lipids. During evolution from a prototype gene, novel functions may be acquired by duplicated daughter genes; for Rab proteins, this can be tested by location, which is specifically related to the function of each Rab. We have found an example of two rab genes in Trypanosoma brucei (trab genes) that clearly arose by tandem duplication, being highly related to each other and remaining juxtaposed in the genome, whose products have dramatically different subcellular locations, indicative of discrete functions. These two trab genes, isolated on a single genomic clone, are separated by a short intervening sequence and are in a head-to-tail orientation. The nucleotide sequences of the open reading frames and intervening sequence were determined and show that the genes are paralogues, probably arising from an ancient tandem duplication. Both genes are most homologous to ypt1 and sec4 in the Saccharomyces cerevisiae genome, while phylogenetic reconstruction indicates that although they have clearly diverged, the proteins are more closely related to each other than to other Rab protein sequences available in the data base. Immunofluorescence microscopy, using antibodies raised against the recombinant Trab proteins, clearly demonstrates that the native Trab proteins have completely distinct subcellular locations in the trypanosome. Trab1p is present in a widespread reticular location similar to BiP, suggesting an endoplasmic reticulum location, while Trab7p is observed in a discrete structure adjacent to the kinetoplast. Most interestingly, the Trab7p-positive compartment also appears to divide at the same time, or just prior to, the kinetoplast, i.e. early in mitosis, suggestive of association with structures in the flagellar pocket region. An estimate of the divergence time indicates that the trab1/trab7 duplication occurred approximately 100 million years ago, and therefore, the persistence of this pair suggests an essential role in the survival of T. brucei.
小G蛋白的Ras超家族通过GTP水解及伴随的构象变化来调控单向细胞过程，而这些又受到多种辅助因子的调节。Rab亚家族成员对于装载有运输蛋白和脂质的细胞内小泡的正确靶向和融合很重要。在从原型基因进化的过程中，复制的子代基因可能会获得新功能；对于Rab蛋白来说，可以通过定位来验证这一点，因为定位与每个Rab的功能密切相关。我们在布氏锥虫中发现了两个rab基因（trab基因）的例子，它们显然是通过串联重复产生的，彼此高度相关且在基因组中相邻排列，其产物具有截然不同的亚细胞定位，表明功能不同。这两个trab基因在单个基因组克隆上分离，由一段短的间隔序列隔开，且呈头对头方向。测定了开放阅读框和间隔序列的核苷酸序列，结果表明这两个基因是旁系同源物，可能源于古代的串联重复。这两个基因与酿酒酵母基因组中的ypt1和sec4最为同源，而系统发育重建表明，尽管它们明显分化，但这两种蛋白质彼此之间的关系比与数据库中其他可用的Rab蛋白序列更为密切。使用针对重组Trab蛋白产生的抗体进行免疫荧光显微镜观察，清楚地表明天然Trab蛋白在锥虫中有完全不同的亚细胞定位。Trab1p存在于类似于BiP的广泛网状位置，提示其定位于内质网，而Trab7p则在靠近动基体的离散结构中观察到。最有趣的是，Trab7p阳性区室似乎也在动基体分裂的同时或之前分裂，即在有丝分裂早期，提示其与鞭毛袋区域的结构有关。对分化时间的估计表明，trab1/trab7重复发生在大约1亿年前，因此，这一对基因的持续存在表明它们在布氏锥虫的生存中起着至关重要的作用。
