Fig. 1. alignment of LmRAB7 (using default parameters) with other characterised Rab7s (and TbRAB7) illustrating conservation of GTP/GDP binding domains (G1–G4), the GAP binding domain (Eff) and the prenylation motif (CXC). Note the kinetoplastid specific insertion, LmRAB7 amino acids 143–162. Homo sapiens (P51149); Rattus norvegicus (P09527); Drosophila melanogaster (076742); Arabidosis thaliana (004157); Glycine max (Q43463); Saccharomyces pombe Ypt, Rab7 homologue (094655); S. cerevisiae Ypt, Rab7 homologue (P32939); L. major (Q9N2P5); T. cruzi (Q9XZK0); T. brucei (TRYP9.0.000912_8); L. major Rab1, LmRAB1 (Q25324).

Fig. 2. An artificially rooted phenogram-like tree diagram created from a maximum parsimony analysis of the edited alignment in Fig. 1. LmRAB1 is the designated outgroup. The tree groups LmRAB7, TcRAB7 and TbRAB7 as divergent Rab7s, distinct from higher multi-cellular organism Rab7s and yeast Ypt7. Bootstrap values from 100 replicates are shown. Hs, H. sapiens; Rn, R. norvegicus; Dm, D. melanogaster; At, A. thaliana; Gm, G. max; Sp, S. pombe Ypt, Rab7 homologue; Sc, S. cerevisiae Ypt, Rab7 homologue; Lm, L. major; Tc, T. cruzi; Tb, T. brucei; LmRAB1-outgroup. Accession numbers as in Fig. 1.

Fig. 3. (A) GTP overlay assay of SDS-PAGE fractionated, blotted and renatured recombinant LmRAB7. Recombinant LmRAB7 (35 kDa) bound [³²P]-GTP (control). However, [³²P]-GTP binding was competed out by an excess (1 mM) of GTP (GTP) or GDP (GDP), but not by 1 mM ATP (ATP), indicating that LmRAB7 is a guanine-specific nucleotide binding protein. (B) Expression of LmRAB7 and LmexRAB7 through the Leishmania life cycle. Immunoblotting using polyclonal anti-LmRAB7 identified L. major LmRAB7 and L. mexicana LmexRAB7 as 25 kDa proteins that are expressed in procyclic (P) and metacyclic (M) promastigotes, and in lesion or axenic amastigotes (A) of L. major and L. mexicana, respectively. Using the defined lifecycle stages of L. mexicana, it is clear by comparison with the LmexNMT loading control that LmexRAB7 is expressed at similar levels in procyclics and amastigotes, but is reduced in quiescent metacyclics.

Fig. 4. Immuno-localisation of LmRAB7 in L. major promastigotes (A) and intra-macrophage amastigotes (B). In promastigotes, ConA (red) uptake was utilised to visualise the endocytic network (Ai) and cells were co-stained for LmRAB7 (green) (Aii). DAPI (blue) was used to visualise the nucleus and kinetoplast, and the images overlayed either with (Aiv) or without (Aiii) a DIC image. In promastigotes, L. major LmRAB7 occupies compartments primarily located between the nucleus and kinetoplast. ConA and LmRAB7 partially co-localise (Aiii), indicating that LmRAB7 occupies an accessible endocytic compartment, presumably late endosomal/lysosomal. Intra-macrophage amastigotes were co-stained with anti-LmRAB7 and DAPI and overlayed with a phase contrast image (Bi). Magnification of the amastigotes (Bii) shows that LmRAB7 resides in a compartment, again presumably late endosomal/lysosomal, near the kinetoplast. Scale BAR=10 μm.

Fig. 5. LmexRAB7 is found close to and on CPB-labelled structures (megasomes/lysosomes): Immuno-electron microscopy of L. mexicana axenic amastigotes. In the four examples shown, the cysteine proteinase, CPB (15 nm gold particles, large arrowheads) localises to electron dense compartments presumed to be megasomes, and LmexRAB7 (10 nm gold particles, arrows) is found close to and overlapping these structures, indicating a role in terminal stages of the endocytic pathway. The presence of additional reactivity in adjacent areas of the cell is probably due to the presence of the two antigens in late endosomes, which are not always easily visualised using this technique. Scale BAR=100 nm. Positions of the nuclei are indicated.