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First published online 26 April 2005
doi: 10.1242/jcs.02327

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The membrane-bound histidine acid phosphatase TbMBAP1 is essential for endocytosis and membrane recycling in Trypanosoma brucei

Markus Engstler^1,*, Frank Weise^2,, Karoline Bopp^1,, Christoph G. Grünfelder², Mark Günzel¹, Niko Heddergott¹ and Peter Overath^2,

¹ Ludwig-Maximilians-Universität, Department Biologie I, Genetik, Maria-Ward-Strasse 1a, München, 80638, Germany
² Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, Tübingen, 72076, Germany

View larger version (60K): [in a new window]   Fig. 1. Schematic view of the posterior part of the bloodstream stage of T. brucei. CCV I, class I clathrin-coated vesicles; CCV II, class II clathrin-coated vesicles; FP, flagellar pocket; EE, early endosomes; RE, recycling endosomes; LE; late endosomes; EXC, exocytic carrier vesicles; L, lysosome; ER, endoplasmic reticulum; G, Golgi complex; N, nucleus; PM, plasma membrane; F, flagellum. See text for explanations. Modified from Engstler et al. (Engstler et al., 2004).

View larger version (60K): [in a new window]   Fig. 2. (A) Schematic representation of the TbMBAP1 protein of T. brucei. The putative signal sequence is highlighted in red, the catalytically active regions appear in blue and the predicted transmembrane region in grey. The C-terminal amino acids recognized by an anti-peptide antibody appear as a black box. Green lines denote potential N-glycosylation sites. The PFAM domain PF00328 (histidine acid phosphatase) is highlighted in yellow. Numbers indicate amino acid positions. (B) Predicted structure of the TbMBAP1 core domain (amino acids 32-326). The coordinates are based on three homology models that were independently generated with CPHmodels-2.0 (http://www.cbs.dtu.dk/services/CPHmodels/), 3D-JIGSAW-2.0 (http://www.bmm.icnet.uk/servers/3djigsaw/) and SDSC1 (http://cl.sdsc.edu/hm.html). The three sets of coordinates were analysed, optimized and visualized with the Deep View software (http://www.expasy.org/spdbv/). The images show a ribbon representation of the integrated model. Helices appear in yellow and sheets in blue. The two catalytically important histidine residues are shown in red and putative N-glycosylation sites in green. Numbers indicate amino acid positions. (C) Monoclonal antibody mAT502 binds to TbMBAP1. An aliquot of a detergent extract from the total membrane fraction of T. brucei bloodstream forms was immunoprecipitated using the mouse monoclonal antibody mAT502. Samples of the precipitate (lane 3) and the supernatant (lane 2) of the immunoprecipitation as well as the detergent extract (lane 1), each corresponding to the equivalent of 2x106 cells, were subjected to immunoblotting. The blot was probed with rabbit anti-TbMBAP1 and horseradish peroxidase coupled secondary antibodies. The molecular mass of standard proteins in kDa is indicated.

View larger version (93K): [in a new window]   Fig. 3. Analysis of the cellular distribution of TbMBAP1 in bloodstream stage trypanosomes. (A) Quantitative colocalization analysis of TbMBAP1 and organelle marker proteins. Nuclear and mitochondrial DNA was stained with DAPI (blue). TbMBAP1 was visualized with ZenonTM-Alexa Fluor 488-labeled anti-TbMBAP1-specific rabbit IgG (middle panel, green). The respective marker proteins were detected with specific antibodies and Alexa Fluor 594 secondary antibodies (right panel, red). The generation of the antibodies against marker molecules has been described previously: clathrin heavy chain (Morgan et al., 2001); RAB5 (Field et al., 1998); RAB11 (Jeffries et al., 2001); p67 (Kelley et al., 1999); BiP (Bangs et al., 1993). The merged colour channels are shown in the left panels. All images are representative examples from a 3D quantitative colocalization analysis (Imaris Surpass Colocalisation software, Bitplane, CH). Sampling numbers (n) are given in the left panels. The numbers in the middle panels indicate the percentage of TbMBAP1 colocalizing with the respective marker proteins, and in the right panel the percentage colocalization of markers with TbMBAP1 is given [for details see Materials and methods and Engstler et al. (Engstler et al., 2004)]. (B) Colocalization of TbMBAP1 with endocytosed VSG. Cells were labelled on ice with sulfo-NHS-SS-biotin and AMCA-sulfo-NHS. Following endocytosis to the steady state (5 minutes at 37°C), cell surface biotin was removed with glutathione. Red colour: Endocytosed VSGbiotin detected with Alexa FluorTM 594 conjugated Streptavidin. VSGAMCA fluorescence (blue) is exclusively visible on the cell surface because the intracellular fluorescence is quenched. TbMBAP1 visualized with anti-TbMBAP1-specific rabbit antiserum and Alexa FluorTM 488-conjugated goat anti-rabbit IgG is in green. The representative image (left panel, top) shows a maximum intensity projection of the corresponding deconvolved 3-channel 3D data set [see Materials and Methods and references (Engstler et al., 2004; Grünfelder et al., 2003)]. Bottom left and right panels: for better visualization, a morphological gradient segmentation was applied to the VSG and MBAP channels. Right panel: enlarged view of the endocytic compartment showing the fluorescence of TbMBAP1, VSGbiotin and the merged image. The filled arrowhead points to regions in which TbMBAP1 does not colocalize with VSGbiotin, the reverse situation is marked by an open arrowhead. N, nucleus; FP, flagellar pocket. Bars: 3 µm. (C-E) Detection of TbMBAP1 in bloodstream form trypanosomes by immunoelectron microscopy. Cryosections were labelled with anti-TbMBAP1 antibodies and PAG-6 (C,D). For improved visibility the gold grains of the images were digitally enlarged. The protein can be detected in the Golgi complex (G), on its cis-side in the adjacent budding zone (BZ, in C), and in many profiles of endosomal cisternae (ECs). Specific labelling is also observed on disk-like exocytic carrier vesicles (EXC), which are abundant near the flagellar pocket (FP in D). The inset in D shows top views of EXCs in the process of fusion with the flagellar pocket membrane. The arrow in C points to a type II clathrin-coated bud at the rim of an endosomal cisterna. (E) In the control with pre-immune serum, EXCs are not labelled. cEC, circular endosomal profile; L, lysosome; MVB, multi-vesicular body; SC, surface coat; FL, flagellum.

View larger version (32K): [in a new window]   Fig. 4. Moderate overexpression of TbMBAP1. (A) The TbMBAP1 gene was expressed under the control of a constitutive (K) or tetracycline-inducible (Ti) procyclin promoter. Quantification of TbMBAP1 on immunoblots shows a moderate overexpression of three to fourfold compared to the wild type (wt). The red colour indicates the TbMBAP1 protein detected with anti-TbMBAP1-specific rabbit antiserum and IRDye 800 second reagent. As a loading control, a mouse anti-HSP60 antibody was detected with Alexa FluorTM 680-conjugated second antibody. Fluorescence quantification was done with the LiCor Odyssey system (see Materials and Methods). (B) Detection of overexpressed TbMBAP1 in the flagellar pocket. Cells were surface-labelled with AMCA-sulfo-NHS (blue). Detection of TbMBAP1 (green) and image processing were as in Fig. 3A. The lysosomal membrane protein p67 was visualized with a specific monoclonal antibody and Alexa-594-conjugated second antibody. While in wild-type cells TbMBAP fluorescence is absent from the flagellar pocket (see Fig. 3A), the protein can be detected in flagellar pocket (FP) membrane upon fourfold overexpression of the protein. The overall localization of TbMBAP1 between flagellar pocket and nucleus (N) is not affected.

View larger version (40K): [in a new window]   Fig. 5. Inducible, strong overexpression of TbMBAP1. A cell line expressing TbMBAP1 under the control of the tetracycline-inducible T7 promoter was grown in the absence of tetracycline for 4 days, which reduced the enzyme to the wild-type level (normalized to 1 at time 0 hours in A and B). The culture was then induced with tetracycline for 24 hours, subsequently deprived of the inducer for 24 hours and finally grown for 12 days in the presence of tetracycline. (A) Immunoblot showing the increase in TbMBAP1 within 24 hours (left) and 12 days (right) of induction, which was quantified relative to the uninduced cells as indicated by the numbers above the blot. Asterisk: cells were grown for 12 days in the absence of tetracycline. TbHSP60 was used as a loading control. (B) Relative TbMBAP1 activity at various time points during the same experiment. Asterisk: cells grown for 24 hours without tetracycline. (C) 3D-Fluorescence microscopy of cells before (0 hours) and after 3, 5 and 20 hours of induction. The cell surface is visualized by AMCA-sulfo-NHS staining (grey), TbMBAP1 by anti-C-terminus antibodies and Alexa FluorTM 594-labeled secondary antibodies (red). For each time point one representative cell is shown as an isosurface rendered, deconvolved 3D image. The white asterisks indicate the location of the flagellar pocket.

View larger version (52K): [in a new window]   Fig. 6. Tetracycline-inducible knockdown by RNAi shows that TbMBAP1 is essential for growth of T. brucei bloodstream cells. (A) Growth in the presence (black circles), absence (white circles) of TbMBAP1-dsRNA. (B) Immunoblot showing the decrease in TbMBAP1 within 20 hours of RNAi. HSP60 was used as a loading control. (C) 3D-Fluorescence microscopy of cells before (0) and after 4, 7 and 12 hours of induction. Upper panel: The cell surface is visualized by AMCA-sulfo-NHS staining (grey), TbMBAP1 by anti-C-terminus antibodies and Alexa FluorTM 594-labeled secondary antibodies (red), and endosomes (green) by the autofluorescence of the marker protein EP:GFP (Engstler and Boshart, 2004). The yellow colour indicates colocalization of GFP and TbMBAP1. The arrows point to the flagellar pocket. In the lower panels only TbMBAP1 is shown. For each time point, images of representative cells were chosen. For the ease of visualization the deconvolved 3D data were processed by isosurface rendering.

View larger version (58K): [in a new window]   Fig. 7. Comparative phenotypic analysis of inducible RNAi against TbMBAP1, clathrin heavy chain and RAB11. (A) Kinetics of endocytosis of the fluid-phase marker Alexa FluorTM 488 dextran in the presence (black circles) or absence (white circles) of tetracycline. Internalization is expressed as the change in relative fluorescence intensity/cell. Analyses were done 6 hours (TbMBAP1, TbCLH) and 10 hours (RAB11) post-induction of RNAi. (B) Localization of the endosomal reporter protein EP:GFP in wild-type cells and after induction of RNAi. The cell surface was stained with AMCA-sulfo-NHS (blue) as described in the legend to Fig. 3B. EP:GFP fluorescence is shown in yellow. The arrows indicate the flagellar pocket. (C) Single frames of time-lapse movies of live cells treated with the plasma membrane dye FM®2-10. Cells were incubated with FM®2-10 on ice for 5 minutes and subsequently warmed to 37°C for 10 minutes. While the control cells (left) and TbRAB11-depleted trypanosomes (right) internalize the fluorescent plasma membrane by endocytosis, the TbMBAP1RNAi (mid left) and TbCLHRNAi cells (mid right) do not show staining of intracellular membranes. The inset in the right image demonstrates that TbRAB11RNAi cells are unable to exocytose internalized membrane even after incubation in the absence of FM®2-10 for 1 hours at 37°C (for details see results section). Arrows indicate the flagellar pocket.

View larger version (26K): [in a new window]   Fig. 8. The expression of TbMBAP1 is developmentally regulated. (A) Immunoblot showing the decrease in TbMBAP1 during development from the bloodstream stage (BSF) to the procyclic insect stage (PCF). Developmental transformation was induced in vitro by the addition of cis-aconitate and a change in culture temperature from 37°C to 27°C. Note that the downregulation of TbMBAP1 is accompanied by a marked mobility-shift of the protein after 72 hours. TbHSP60 was used as a loading control. (B) Relative TbMBAP1 activity at various time points during differentiation. (C) Kinetics of endocytosis of the fluid-phase marker Alexa FluorTM 488 dextran during development to the procyclic insect stage. At the time points indicated, cells were incubated with fluorescent dextran for 15 minutes at 27°C. Internalization is expressed as the change in relative fluorescence intensity/cell.

View larger version (42K): [in a new window]   Fig. 9. Schematic representation of membrane flow between endosomes and cell surface. Membrane areas are drawn as spheres and are approximately to scale (based on Grünfelder et al., 2002). The green colour indicates the localization of the EP:GFP reporter. Dark arrows indicate membrane flow and light grey arrows symbolize the impaired flow of membrane caused by RNAi. F, flagellum; FP, flagellar pocket; E, endosomes. The dotted lines mark membrane compartments that are connected to either the pellicular microtubule cytoskeleton or the flagellar axoneme. In wild-type cells endo- and exocytic traffic are balanced. The EP:GFP reporter is shuttled between endosomes and the flagellar pocket. In trypanosomes depleted of clathrin heavy chain (TbCLH) endocytosis halts while exocytosis continues. This results in a specific enlargement of the flagellar pocket. The EP:GFP reporter is found exclusively in the flagellar pocket. After downregulation of TbRAB11 endocytosis continues while exocytosis is blocked, resulting in the clearance of EP:GFP from the flagellar pocket. Knockdown of TbMBAP1 affects both endocytosis and exocytosis.