Cell-cycle and developmental regulation of TbRAB31 localisation, a GTP-locked Rab protein from Trypanosoma brucei

Rab proteins are small GTPases that control the direction and timing of ves icle fusion during intracellular trafficking between membraneous compartmen ts. Genome sequencing and EST analysis of Trypanosoma brucei indicates that the trypanosome Rab (TbRAB) gene family, and hence complexity of intracell ular transport pathways, is intermediate between Saccharomyces cerevisae an d mammals. TbRAB31 is a constitutively expressed T. brucei Rab protein (for merly Trab7p) and is the product of one of two closely linked TbRAB genes, the other being TbRAB2 (TbRab2p, in: Field H, Ali BRS, Sherwin T, Gull K: C roft SL, Field MC. TbRab2p, a marker for the endoplasmic reticulum of Trypa nosoma brucei, localises to the ERGIC in mammalian cells. J Cell Sci 1999.1 12.147-156). involved in ER to Golgi transport. TbRAB31 has high homology t o members of the Sec4/Ypt1 subfamily of Rab proteins from S. cerevisiae and to Rab13 and Rab11 from higher eukaryotes. Recombinant TbRAB31 binds GTP b ut, unusually for a Rab protein, has undetectable GTPase activity resulting in a constitutively GTP-bound protein. Antibodies against TbRAB31 recognis e a discrete structure located between the kinetoplast and nucleus in inter phase procyclic cells: by contrast the structure is morphologically more co mplex in bloodstream form (BSF) parasites, consisting of at least two foci. TbRAB31 behaviour was also studied during the cell cycle; TbRAB31 always l ocalised to a discrete structure that duplicated very early in mitosis and relocated to daughter cells in a coordinate manner with the basal body and kinetoplast, suggesting the involvement of microtubules. Additional evidenc e suggests that TbRAB31 localises to the trypanosome Golgi complex. Firstly , the interphase position of TbRAB31 is consistent with a Golgi location. S econdly, the TbRAB31 structure is also recognised by cross-reacting antibod ies to mammalian beta-coatomer protein (beta-COP), which localises to the G olgi in mammalian cells. Thirdly, the fluorescent ceramide analogue, BODIPY -TR-ceramide, a reliable marker of the mammalian Golgi apparatus, exhibited overlapping distribution with TbRAB31. The location of BODIPY-TR-ceramide was confirmed at the trypanosome Golgi by histochemistry with diaminobenzid ine and electron microscopy. (C) 2000 Elsevier Science B.V. All rights rese rved.