TANDEM DUPLICATION OF RAB GENES FOLLOWED BY SEQUENCE DIVERGENCE AND ACQUISITION OF DISTINCT FUNCTIONS IN TRYPANOSOMA-BRUCEI

The Ras superfamily of small G proteins governs unidirectional cellula r 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 ma y 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 hi ghly related to each other and remaining juxtaposed in the genome, who se products have dramatically different subcellular locations, indicat ive of discrete functions. These two trab genes, isolated on a single genomic clone, are separated by a short intervening sequence and are i n a head-to-tail orientation, The nucleotide sequences of the open rea ding frames and intervening sequence were determined and show that the genes are paralogues, probably arising from an ancient tandem duplica tion, Both genes are most homologous to ypt1 and sec4 in the Saccharom yces cerevisiae genome, while phylogenetic reconstruction indicates th at 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 T rab7p is observed in a discrete structure adjacent to the kinetoplast, Most interestingly, the Trab7p-positive compartment also appears to d ivide 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 similar to 100 million years ago, and therefore, the persistence of this pair suggests an essential role in the survival of T. brucei.