Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - (A) Phylogenetic analysis of the T. brucei ARF proteins and other eukaryotic relatives (PHYLIP, Cluster algorithm based on CLUSTALW alignment), with the putative T. brucei GTPase RAN (Tb927.3.1120) as an outgroup. (B) Alignment of the T. brucei ARF proteins with other eukaryotic relatives. The Switch I and II effector domains are shown in blue and grey shading, respectively. The motif MXXE (blue text) is found in Golgi-localizing ARFs (Honda et al., 2005). A conserved ARF6 motif (Al-Awar et al., 2000, red text) is absent from T. brucei and S. cerevisiae ARF sequences. Residues 2, 31 and 71 were targets for sitedirected mutagenesis and are shown in green text. Tb, Trypanosoma brucei. Tc, Trypanosoma cruzi. Lm, Leishmania major. Yeast, Saccharomyces cerevisiae. Xen, Xenopus laevis. Chick, Gallus gallus. Asp, Aspergillus terreus. Accession numbers (GeneDB or SwissProt): TbARF1 (4 tandem identical copies), Tb09.211.4460, Tb09.211.4470, Tb09.211.4480, Tb09.211.4490. TbARF2, Tb09.160.5600. HumanARF1, P32889. TbARF2, Tb09.160.5600. HumanARF6, P26438. YeastARF3, P40994. MouseARF1, BAA13490. RatARF1, NP_071963. XenARF1, BAE94175. ChickARF1, NP_001006352. AspARF1, EAU39010. MouseARF6, BAA13495. RatARF6, AAH91146. ChickARF6, P26990. XenARF6, BAE94177. AspARF6, XP_00121393.
• Supplemental Figure 2 - RNA interference of TbARF1 expression in bloodstream form (BSF) parasites. (A) Growth of the BSF transfectedline 9013/p2T7/ARF1 in the absence (blue Δ) and presence (pink Δ) of tetracycline, monitored over a 5 day time course. (B) Quantitative (Real Time) PCR to measure the number of molecules of TbARF1 RNA transcript per molecule of α-tubulintranscript, in the 9013 BSF parental cell line (wt) and the transfectedcell line 9013/p2T7/ARF1 (RNAi) grown in the absence or presence of tetracycline for up to 16 hours. (C) Nuclei and kinetoplastswere counted in the BSF line as above, in the absence or presence of tetracycline over a 24 hour time course. At least 250 cells were counted per sample. Configurations other than K1N1, K2N1 and K2N2 were classified asabnormal. (D) Abnormal cell morphology was quantified by light microscopy in the BSF line as above over a 24 hour time course following addition of tetracycline. The percentage of cells with an enlarged (4x normal size or greater) flagellarpocket (black Δ) and the percentage of round cells (blue ·) are shown for each time point. At least 250 cells were scored per sample.
• Supplemental Figure 3 - Expression of myc-tagged TbARF1 wild-type and mutant proteins in BSF parasites. (A) Growth of transfectedcell lines 427/pM2ARF1/WT or mutant forms as shown, grown in the absence (blue ♦) and presence (pink ♦) of tetracycline. (B) Total lysatesof cells grown in the absence or presence of tetracycline for 16 hours (or 2 hours formutant Q71L) were immunoblotted(1x 10₇ cells/lane) and probed with anti-mycor anti-BiPto monitor equal sample loading. (C) Expression of myc-tagged TbARF1 wild-type and G2A mutant proteins in the absence of tetracycline (-), presence of tetracycline (T), and presence of both tetracycline and protease inhibitors (T/i). Cells were induced for 8 hours and total lysates(1 x 10₇cells/lane) immunoblottedas above. (D) Subcellularfractionation of cells grown in the absence or presence of tetracycline for 16 hours. C, cytosolicfraction. M, membrane fraction. Immunoblotswere probed with anti-mycor anti-BiPas above. (E) Quantitative (Real Time) PCR to measure the number of molecules of TbARF1 transcript per molecule of α-tubulin, in cells grown in the absence or presence of tetracycline for up to 16 hours.
• Supplemental Figure 4 - Immunofluorescenceassays of cells expressing TbARF1 mutant proteins, grown in the presence of tetracycline for 16 hours (or 2 hours for Q71L mutant). (A) Cells expressing ARF1 proteins were stained with anti-myc(green). (B) Cells expressing ARF1 proteins were stained with antibodies against the lysosomalmarker p67 (green) and the early endosomalmarker Rab5 (red). All cells were co-stained with DAPI. Bar, 5 μm.