Arrows demark where the FAZ originates

Arrows demark where the FAZ originates. of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope GW 441756 [1], which is clearly inconsistent with the mammalian location and function. As T. brucei is usually unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s) of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell GW 441756 cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is usually conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes. strong class=”kwd-title” Keywords: Rab23, flagellum, trafficking, trypanosome, IFT Introduction Rab proteins are Rabbit polyclonal to cox2 important control elements of vesicle transport and related functions [2]. In African trypanosomes there are sixteen Rab-like proteins with various functions in exocytosis, endocytosis and possibly differentiation/life cycle progression [3-5]. Essentially members of the family have similar locations and functions to their mammalian or fungal orthologues where such orthologous associations exist [4,6]. In contrast, amongst the Trypanosoma brucei Rab repertoire is usually Rab23 (TbRab23), which was localized to the inner nuclear envelope using polyclonal antibodies [1]. Significantly, mammalian Rab23 was first recognized in a murine model through genetic interactions with Sonic hedgehog (Shh), a protein crucial to development and differentiation of cell lineages [7,8]. Mammalian Rab23 localizes to the plasma membrane, endosomes, and cytosol and functions as a negative regulator of Shh [9-11]. The Shh pathway is usually metazoan-specific and essential for growth and organ patterning [12,13]. Several components of the pathway localize to primary cilia and intraflagellar transport (IFT) is required for Shh signaling [14-18]. Recent data indicate that Rab23 is essential for ciliogenesis, localizes to cilia in MDCK cells and functions GW 441756 to recycle the Shh pathway receptor, Smoothened (Smo), from the ciliary compartment [19,20]. As Shh signaling is restricted to metazoa, this raises intriguing questions about the possible function of TbRab23 in T. brucei and other non-metazoan organisms and may point towards a divergent function. Given that no Rab protein has been associated with the nucleus in any organism since our earlier publication, and accumulation of evidence for a role of Rab23 at the mammalian flagellum, we sought to re-examine the location of Rab23 in trypanosomes and its phylogenetic relationship with the presence of a flagellum. Using epitope-tagged chimeras of TbRab23 to avoid issues with antisera cross-reactivity, we now find that TbRab23 is usually associated with the flagellum, and not the nucleus as previously reported [1]. Further, Rab23 is only found in flagellated taxa. Importantly our data are consistent with the studies in mammalian cells and indicates that this function of Rab23 is likely conserved across the eukaryotes. 2. Materials and methods 2.1. Bioinformatics Genomes were chosen for sampling based on three main criteria: i) proposed position in the evolutionary tree of eukaryotes [21] ensuring adequate representation of major supergroups, ii) completeness of the sequenced genome, and iii) the availability of a carefully annotated protein database. BLAST searches were conducted initially using TbRab23 (Tb10.6k15.1990), TbRab28 (Tb927.6.3040), TbRab7 (Tb09.211.2330), HsRab23 (“type”:”entrez-protein”,”attrs”:”text”:”NP_057361.3″,”term_id”:”34485714″,”term_text”:”NP_057361.3″NP_057361.3), and HsRab28 (“type”:”entrez-protein”,”attrs”:”text”:”CAA64364.1″,”term_id”:”1154852″,”term_text”:”CAA64364.1″CAA64364.1) as queries [22]. As GTPases are very highly conserved, e values of less than e-20 were used as an initial cutoff. The top two hits were then reciprocally used as queries for a BLAST search against T. brucei and Homo sapiens sequence databases. Only those that retrieved the query sequence GW 441756 were assigned as.