The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet–Biedl syndrome, acting
at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking
events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated
model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which
has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following
co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation
of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction
in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an
anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between
ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation
of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of
BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate
that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the
BBSome