Journal of Cell Science RESEARCH ARTICLE The bi-lobe-associated LRRP1 regulates Ran activity in Trypanosoma brucei Anaı ¨s Brasseur 1,` , Shima Bayat 1,` , Xiu Ling Chua 1 , Yu Zhang 1 , Qing Zhou 3, *, Boon Chuan Low 2 and Cynthia Y. He 1,§ ABSTRACT Cilia and flagella are conserved eukaryotic organelles important for motility and sensory. The RanGTPase, best known for nucleocytoplasmic transport functions, may also play a role in protein trafficking into the specialized flagellar/ciliary compartments, although the regulatory mechanisms controlling Ran activity at the flagellum remain unclear. The unicellular parasite Trypanosoma brucei contains a single flagellum necessary for cell movement, division and morphogenesis. Correct flagellum functions require flagellar attachment to the cell body, which is mediated by a specialized flagellum attachment zone (FAZ) complex that is assembled together with the flagellum during the cell cycle. We have previously identified the leucine-rich-repeat protein 1 LRRP1 on a bi-lobe structure at the proximal base of flagellum and FAZ. LRRP1 is essential for bi-lobe and FAZ biogenesis, consequently affecting flagellum-driven cell motility and division. Here, we show that LRRP1 forms a complex with Ran and a Ran-binding protein, and regulates Ran–GTP hydrolysis in T. brucei. In addition to mitotic inhibition, depletion of Ran inhibits FAZ assembly in T. brucei, supporting the presence of a conserved mechanism that involves Ran in the regulation of flagellum functions in an early divergent eukaryote. KEY WORDS: Bi-lobe, Flagellum, LRRP1, RanBP, Ran INTRODUCTION Protein trafficking into the membrane-bound flagellum or cilium is tightly controlled by polarized vesicular trafficking as well as a diffusion barrier located at the base of the structure (Hsiao et al., 2012), creating a privileged compartment crucial for flagellar biogenesis and functions. In recent years, several studies have described a role of the small GTPase Ran – best known for its biological function in nucleocytoplasmic protein transport (Sazer and Dasso, 2000) – in the regulation of ciliary protein targeting in various eukaryotes (Dishinger et al., 2010; Fan et al., 2011; Ludington et al., 2013). Whereas the regulation of Ran activity at the nucleus is relatively well-understood, how Ran activity is regulated at the ciliary base is not yet clear. In Trypanosoma brucei, the etiological agent for human african trypanosomiasis (HAT; also known as sleeping sickness), a single flagellum is present in each parasite cell, required for cell locomotion, immune evasion, cell division, organelle positioning and cell length regulation (Absalon et al., 2008; Absalon et al., 2007; Broadhead et al., 2006; Engstler et al., 2007; Kohl et al., 2003; Ralston et al., 2006). The normal function of T. brucei flagellum requires flagellum attachment to the cell body, through the flagellum attachment zone (FAZ), a unique feature found in T. brucei and related trypanosomatid parasites (Vaughan, 2010). Disruption of flagellum attachment in T. brucei leads to defects in flagellum-driven cell motility and cell division as well as changes in cell morphology, often resulting in cell death (LaCount et al., 2002; Sun et al., 2013; Vaughan et al., 2008; Woods et al., 2013). Little, however, is known about the protein composition of the FAZ complex and how it is assembled together with the flagellum. A bi-lobe structure is present at the proximal base of the single flagellum, partially overlapping with the proximal tip of the FAZ (Morriswood et al., 2012; Shi et al., 2008) and adjacent to the single Golgi complex (He et al., 2005). Previously thought to be required for Golgi duplication, recent studies suggest a more fundamental role of the bi-lobe in FAZ assembly that, consequently, affects flagellum functions in organelle inheritance, cell division and cell morphogenesis (Zhou et al., 2010; Zhou et al., 2011). Depletion of the bi-lobe proteins Centrin2 (gene accession number Tb927.8.1080) and LRRP1, both leads to defects in FAZ assembly, flagellum inheritance, flagellum-driven cell motility and cell division (Shi et al., 2008; Zhou et al., 2010). To better understand the bi-lobe and the molecular function of LRRP1 and to identify LRRP1-interacting proteins, we carried out yeast-two-hybrid screens. The results showed that LRRP1 forms a complex with Ran GTPase and a previously uncharacterized Ran-binding protein. Further functional characterization suggested a role of LRRP1 as a Ran regulator specific for flagellar functions. RESULTS Yeast-two-hybrid screening for LRRP1-interacting proteins T. brucei LRRP1 has previously been identified in a comparative proteomics screening for flagellum-associated proteins (Zhou et al., 2010). The 713-aa protein lacks sequence homology to other organisms but is conserved in trypanosomatids. LRRP1 is stably associated with the bi-lobe structure in T. brucei, shown by stable expression of YFP-tagged LRRP1 or by immunostaining with antibody against LRRP1 (Zhou et al., 2010). LRRP1 comprises an N-terminal leucine-rich repeat (LRR) domain followed by a 50-aa- long coiled-coil region within the C-terminal part (Fig. 1A). LRR 1 Department of Biological Sciences, Centre for BioImaging Sciences, National University of Singapore, 14 Science Drive 4, S1A #06-04, Singapore 117543, Singapore. 2 Singapore Mechanobiology Institute, National University of Singapore, T-Lab, #10-01, 5A Engineering Drive 1, Singapore 117411, Singapore. 3 University of Texas Medical School. *Present address: Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX 77030, USA. ` These authors contributed equally to this work § Author for correspondence (dbshyc@nus.edu.sg) Received 13 December 2013; Accepted 14 August 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4846–4856 doi:10.1242/jcs.148015 4846