Rab23 is a flagellar protein in Trypanosoma brucei

BMC Research Notes

Mark C Field¹  Jennifer H Lumb¹ 

[1] Cambridge Institute for Medical Research, MRC/Wellcome Trust building, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0XY, UK

关键词: IFT;    trypanosome;    trafficking;    flagellum;    Rab23;   

Others  :  1167465
DOI  :  10.1186/1756-0500-4-190

 received in 2010-12-01, accepted in 2011-06-15,  发布年份 2011

【 摘 要 】

Background

Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope [1], which is clearly inconsistent with the mammalian location and function. As T. brucei is 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 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 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.

【 授权许可】

   
2011 Lumb et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Dhir V, Field MC: TbRAB23; a nuclear-associated Rab protein from Trypanosoma brucei. Mol Biochem Parasitol 2004, 136:297-301.
• [2]Stenmark H: Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 2009, 10:513-25.
• [3]Ackers JP, Dhir V, Field MC: A bioinformatic analysis of the RAB genes of Trypanosoma brucei. Mol Biochem Parasitol 2005, 141:89-97.
• [4]Field MC, Carrington M: The trypanosome flagellar pocket. Nat Rev Microbiol 2009, 7:775-86.
• [5]Natesan SK, Peacock L, Leung KF, Matthews KR, Gibson W, Field MC: The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity. PLoS One 2009, 4:7217.
• [6]Brighouse A, Dacks JB, Field MC: Rab protein evolution and the history of the eukaryotic endomembrane system. Cell Mol Life Sci 2010, 67:3449-56.
• [7]Eggenschwiler JT, Espinoza E, Anderson KV: Rab23 is an essential negative regulator of the mouse Sonic hedgehog signaling pathway. Nature 2001, 412:194-8.
• [8]Hooper JE, Scott MP: Communicating with Hedgehogs. Nat Rev Mol Cell Biol 2005, 6:306-17.
• [9]Evans TM, Simpson F, Parton RG, Wicking C: Characterization of Rab23, a negative regulator of sonic hedgehog signaling. Methods Enzymol 2005, 403:759-77.
• [10]Guo A, Wang T, Ng EL, Aulia S, Chong KH, Teng FY, Wang Y, Tang BL: Open brain gene product Rab23: expression pattern in the adult mouse brain and functional characterization. J Neurosci Res 2006, 83:1118-27.
• [11]Eggenschwiler JT, Bulgakov OV, Qin J, Li T, Anderson KV: Mouse Rab23 regulates hedgehog signaling from smoothened to Gli proteins. Dev Biol 2006, 290:1-12.
• [12]McMahon AP, Ingham PW, Tabin CJ: Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol 2003, 53:1-114.
• [13]Varjosalo M, Taipale J: Hedgehog: functions and mechanisms. Genes Dev 2008, 22:2454-72.
• [14]Haycraft CJ, Banizs B, Aydin-Son Y, Zhang Q, Michaud EJ, Yoder BK: Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet 2005, 1:e53.
• [15]Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DY, Reiter JF: Vertebrate Smoothened functions at the primary cilium. Nature 2005, 437:1018-21.
• [16]Rohatgi R, Milenkovic L, Scott MP: Patched1 regulates hedgehog signaling at the primary cilium. Science 2007, 317:372-6.
• [17]Kovacs JJ, Whalen EJ, Liu R, Xiao K, Kim J, Chen M, Wang J, Chen W, Lefkowitz RJ: Beta-arrestin-mediated localization of smoothened to the primary cilium. Science 2008, 320:1777-81.
• [18]Tran PV, Haycraft CJ, Besschetnova TY, Turbe-Doan A, Stottmann RW, Herron BJ, Chesebro AL, Qiu H, Scherz PJ, Shah JV, Yoder BK, Beier DR: THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia. Nat Genet 2008, 40:403-10.
• [19]Yoshimura S, Egerer J, Fuchs E, Haas AK, Barr FA: Functional dissection of Rab GTPases involved in primary cilium formation. J Cell Biol 2007, 178:363-9.
• [20]Boehlke C, Bashkurov M, Buescher A, Krick T, John AK, Nitschke R, Walz G, Kuehn EW: Differential role of Rab proteins in ciliary trafficking: Rab23 regulates smoothened levels. J Cell Sci 2010, 123:1460-7.
• [21]Roger AJ: The real 'kingdoms' of eukaryotes. Curr Biol 2004, 14:R693-6.
• [22]Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-402.
• [23]Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal × version 2.0. Bioinformatics 2007, 23:2947-8.
• [24]Maddison WP, Maddison DR: Interactive analysis of phylogeny and character evolution using the computer program MacClade. Folia Primatol 1989, 53:190-202.
• [25]Abascal F, Zardoya R, Posada D: ProtTest: Selection of best-fit models of protein evolution. Bioinformatics 2005, 21(9):2104-2105.
• [26]Whelan S, Goldman N: A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol 2001, 18:691-699.
• [27]Field H, Field MC: Tandem duplication of rab genes followed by sequence divergence and acquisition of distinct functions in Trypanosoma brucei. J Biol Chem 1997, 272:10498-505.
• [28]Wirtz E, Leal S, Ochatt C, Cross GA: A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 1999, 99:89-101.
• [29]Bastin P, MacRae TH, Francis SB, Matthews KR, Gull K: Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes. Mol Cell Biol 1999, 19:8191-200.
• [30]Redmond S, Vadivelu J, Field MC: RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei. Mol Biochem Parasitol 2003, 128:115-8.
• [31]Koumandou VL, Natesan SK, Sergeenko T, Field MC: The trypanosome transcriptome is remodelled during differentiation but displays limited responsiveness within life stages. BMC Genomics 2008, 9:298. BioMed Central Full Text
• [32]Sherwin T, Gull K: The cell division cycle of Trypanosoma brucei brucei: timing of event markers and cytoskeletal modulations. Philos Trans R Soc Lond B Biol Sci 1989, 323:573-88.
• [33]Diehl S, Diehl F, El-Sayed NM, Clayton C, Hoheisel JD: Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray. Mol Biochem Parasitol 2002, 123:115-23.
• [34]Adl SM, Simpson AG, Farmer MA, Andersen RA, Anderson OR, Barta JR, Bowser SS, Brugerolle G, Fensome RA, Fredericq S, James TY, Karpov S, Kugrens P, Krug J, Lane CE, Lewis LA, Lodge J, Lynn DH, Mann DG, McCourt RM, Mendoza L, Moestrup O, Mozley-Standridge SE, Nerad TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MF: The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 2005, 52:399-451.
• [35]Chavrier P, Gorvel JP, Stelzer E, Simons K, Gruenberg J, Zerial M: Hypervariable C-terminal domain of rab proteins acts as a targeting signal. Nature 1991, 353:769-72.
• [36]Ali BR, Seabra MC: Targeting of Rab GTPases to cellular membranes. Biochem Soc Trans 2005, 33:652-6.
• [37]Evans TM, Ferguson C, Wainwright BJ, Parton RG, Wicking C: Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway. Traffic 2003, 4:869-84.
• [38]Pereira-Leal JB, Seabra MC: Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 2001, 313:889-901.
• [39]Rout MP, Field MC: Isolation and characterization of subnuclear compartments from Trypanosoma brucei. Identification of a major repetitive nuclear lamina component. J Cell Bio 2001, 276:38261-71.
• [40]Pal A, Hall BS, Jeffries TR, Field MC: Rab5 and Rab11 mediate transferrin and anti-variant surface glycoprotein antibody recycling in Trypanosoma brucei. Biochem J 2003, 374:443-51.
• [41]Absalon S, Blisnick T, Kohl L, Toutirais G, Dore G, Julkowska D, Tavenet A, Bastin P: Intraflagellar transport and functional analysis of genes required for flagellum formation in trypanosomes. Mol Biol Cell 2008, 19:929-44.
• [42]Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson KV: Hedgehog signaling in the mouse requires intraflagellar transport proteins. Nature 2003, 426:83-7.
• [43]Briggs LJ, Davidge JA, Wickstead B, Ginger ML, Gull K: More than one way to build a flagellum: comparative genomics of parasitic protozoa. Curr Biol 2004, 14:R611-2.
• [44]Jekely G, Arendt D: Evolution of intraflagellar transport from coated vesicles and autogenous origin of the eukaryotic cilium. Bioessays 2006, 28:191-8.
• [45]Benchimol M: Trichomonads under Microscopy. Microsc Microanal 2004, 10:528-50.
• [46]Rosenbaum JL, Witman GB: Intraflagellar transport. Nat Rev Mol Cell Biol 2002, 3:813-25.
• [47]Deane JA, Cole DG, Seeley ES, Diener DR, Rosenbaum JL: Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles. Curr Biol 2001, 11:1586-90.
• [48]Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum JL: Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol 1998, 141:993-1008.
• [49]Follit JA, Tuft RA, Fogarty KE, Pazour GJ: The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly. Mol Biol Cell 2006, 17:3781-92.
• [50]Nachury MV, Loktev AV, Zhang Q, Westlake CJ, Peranen J, Merdes A, Slusarski DC, Scheller RH, Bazan JF, Sheffield VC, Jackson PK: A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 2007, 129:1201-13.
• [51]Pazour GJ, Agrin N, Leszyk J, Witman GB: Proteomic analysis of a eukaryotic cilium. J Cell Biol 2005, 170:103-13.
• [52]Broadhead R, Dawe HR, Farr H, Griffiths S, Hart SR, Portman N, Shaw MK, Ginger ML, Gaskell SJ, McKean PG, Gull K: Flagellar motility is required for the viability of the bloodstream trypanosome. Nature 2006, 440:224-7.