Error bars indicate the s.e.m. subunits. These data suggest Rab28 functions in coordinating late endocytic events. Results Rab28 is a novel endocytic protein We examined the role of the orthologue of mammalian Rab28 in membrane transport. offers an attractive system in which to study this Rab protein on account of a streamlined endocytic system coupled to a high level of definition, together with extensive evidence that Rab orthologues maintain broadly similar functions across deep evolutionary RO462005 time (Field and Carrington, 2009; Brighouse et al., 2010). Rab28 (Tb927.6.3040) was initially identified by comprehensive screening of the trypanosome genome for Ras- and Rab-like small GTPases (Berriman et al., 2005; Ackers et al., 2005); Rab28 shares 49% identity and 58% similarity to RAB28 and extensive similarity to orthologues in other taxa, notably within the C-terminal hypervariable domain. Rab28 is widely distributed across the Eukaryota, despite secondary losses resulting in the absence of Rab28 from Plantae, Fungi and Amoebozoa, and therefore Rab28 is dispensable in certain organisms (Lumb and Field, 2011). To examine Rab28 expression in trypanosomes we initially analyzed mRNA levels; quantitative real time PCR (qRT-PCR) confirmed Rab28 transcripts in both bloodstream form (BSF) and procyclic form (PCF) trypanosomes, suggesting a role throughout the life cycle (Fig. 1A). Open in a separate window Fig. 1. Expression of Rab28 and validation of antibodies. (A) mRNA is expressed at similar levels in BSF and PCF cells. Data were RO462005 normalized for RNA input to -tubulin and expression of mRNA in PCF calibrated against expression in BSF. Errors bars show s.e.m. from triplicate RNA extractions (B) Production of TbRab28HA (29 kDa) and TbRab28YFP (51 kDa) proteins of the correct size in trypanosomes was verified by western blot using anti-HA or anti-GFP on transfected BSF cells. (C) Western blot of purified anti-serum (1:1000) against Rab28 on bacterial (10 ng) and cell (107 cells) lysates. Induced denotes bacterial whole cell lysate in which expression of recombinant GST::Rab28 was induced. Rab28 antibodies recognized an antigen of 48 kDa corresponding to GST::Rab28 that was absent in non-induced culture. An antigen (27 kDa) similar to the predicted size of Rab28 (26 kDa) was recognized in BSF and PCF. Presence of endogenous Rab28 in addition to an antigen of 31 kDa corresponding to TbRab28HA (29 kDa) was detected in recombinant Rab28 cells. (D) Immunofluorescence images of wild-type SMB BSF cells (Lister 427) stained with RO462005 antibodies to HA and GFP. Cells were stained with anti-HA and counterstained with Alexa Fluor 568 or with anti-GFP and counterstained with Oregon Green. DNA was visualized with DAPI (blue). Anti-HA and anti-GFP did not cross-react with trypanosome structures, validating the use of these antibodies in subsequent localization experiments. (E) Location of Rab28 during the RPS6KA5 BSF cell cycle. Cells were transfected for expression of N-terminal HA- or YFP-tagged Rab28 and the tagged protein visualized with anti-HA (left panel) or anti-GFP (right panel). DNA in all figures is co-stained with DAPI (blue). 1K1N signifies G1 phase, 2K1N early nuclear S-phase; and 2K2N that the kinetoplast replicates prior to the nucleus. Rab28 locates between the nucleus and kinetoplast. Rab28 localization becomes more extensive at G2. (F) Representative image of endogenous Rab28 in BSF cells. Cells were stained with anti-Rab28 and counterstained with Alexa Fluor 568 (red). (G) Location of Rab28 in PCF cells. HA- and YFP-tagged Rab28 were expressed in PCF cells and visualized with antibodies. Representative images are shown. Scale bars: RO462005 2 m. To determine subcellular location, Rab28 was fused to an N-terminal haemagglutinin (HA) or YFP-epitope tag and ectopically expressed in BSF cells. Production of the respective chimeras, TbRab28HA (31 kDa) and TbRab28YFP (51 kDa), of the correct molecular weight were.