Neighbor-joining tree representing relationships between all known Tetrahymena Rabs, showing bootstrap values above 50%. As in Bright et al. the sequences used for this analysis were trimmed to remove the C-terminal hypervariable domains. Conserved clades are marked with blue boxes. This tree is similar but not identical to the neighbor-joining tree shown in Figure S9 of Bright et al. in that the current tree contains 8 additional Rabs [marked with red circles (divergent) or triangles (conserved)] as reported and named in Saito-Nakano et al.19 In addition, the tree shown here is based on the experimentally verified Rab sequences reported in Saito-Nakano rather than the previously used informatics-based sequences. This change corrected the amino acid sequences of the following Rabs: D31, 5C/D23, D1, D11, 11C, D21, D22, D35, D37, D18. Possibly as a result, several Rabs (marked with blue triangles) appear to be robustly associated with conserved clades, whereas they were previously judged as divergent.

Six examples of localizing Rab-GFP fusions in living immobilized Tetrahymena. (A) Localization to phagosomes that are docked at a site on the cortex, called the cytoproct, where undigested contents are egested. (B) Localization to vesicles traveling along long cytoplasmic microtubules between the cytoproct (posterior) and the oral apparatus (anterior). (C) Localization to endosomes, indentified by co-labeling with an endocytic tracer, FM4-64 (red signal). (D) Localization to the contractile vacuole, an organelle involved in osmotic homeostasis. (E) Localization to Golgi, which in Tetrahymena are linearly arrayed as multiple small stacks near the cell cortex. (F) Localization to the macronuclear envelope. In all parts, the Rab-GFP signal is shown in green. The Tetrahymena cells shown are ∼50 µM in length.