QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 February 2005
doi: 10.1242/jcs.01624

This Article Summary Full Text Full Text (PDF) Supplemental Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Price, H. P. Articles by Smith, D. F. Search for Related Content PubMed PubMed Citation Articles by Price, H. P. Articles by Smith, D. F.

Functional analysis of TbARL1, an N-myristoylated Golgi protein essential for viability in bloodstream trypanosomes

Helen P. Price^*, Chrysoula Panethymitaki, David Goulding and Deborah F. Smith^*,

Wellcome Trust Laboratories for Molecular Parasitology, Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, London, SW7 2AZ, UK

View larger version (85K): [in a new window]   Fig. 1. Alignment of the T. brucei ARF protein family with related sequences. Residues and motifs conserved in ARFs are highlighted in dark grey. These include the GDP/GTP - and ß-phosphate binding site GLDXAGKT, of which the threonine residue is essential for binding to guanine nucleotide exchange factors; WDXGGQ, which interacts with the purine ring of GTP; and NKXD, which binds magnesium and the ß phosphate of GTP. Several Arf-specific residues are found in the majority of the parasite proteins, including N48, W79, R100 and G165. N-myristoylation motifs are indicated by blue shading. Residues essential for binding to GRIP domain proteins are highlighted in yellow.

View larger version (42K): [in a new window]   Fig. 2. Characterization of TbARL1. (A) RT-PCR showing differential expression of ARL1 in procyclic (PCF) and bloodstream (BSF) life cycle stages of T. brucei. Amplification of constitutively expressed Rab4 was used to show equal amounts of cDNA in the reactions. (B) N-myristoylation assay. TbARL1 and TbNMT were expressed as recombinant proteins of 22 kDa and 48.5 kDa respectively in the presence of [3H]myristate, following induction with IPTG (upper panel, Coomassie Blue-stained gel). The radiolabelled products (the NMT-myristoyl CoA binary complex and myristoylated ARL1) were detected by autoradiography (lower panel). The data shown represent one of three independent experiments. (C) Subcellular fractionation of myc-epitope tagged ARL1 wild type (wt) and G2A mutant proteins in BSF T. brucei. Immunoblots were probed with anti-myc antibody to detect ARL1 proteins and anti-BiP antibody to control for equal sample loading. Lanes 1, 2, BSF s427 parental line, cytosolic and membrane fractions respectively; lanes 3, 4, BSF transfected line 427/pM2cCARL1WT grown in tetracycline for 48 hours, cytosolic and membrane fractions, respectively; lanes 5, 6, BSF line 427/pM2cCARL1G2A grown in tetracycline as above, cytosolic and membrane fractions respectively. (D) Immunofluorescence of parasites described in C using anti-myc (red) and anti-Rab1 (green) antibodies. In the bottom panel, cells are co-stained with DAPI (shown in blue). Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 3. RNA interference of TbARL1 expression. (A) Growth of the T. brucei bloodstream form (BSF) transfected line Bp2T7/ARL1 in the absence (open squares) and presence (filled squares) of tetracycline, monitored over a 5 day time course. (B) Northern blots of RNA (10 µg) from procyclic strain 29-13 (PCF), bloodstream parental line 90-13 (BSF) and transfected line Bp2T7/ARL1 grown in the absence (No Tet) and presence (+Tet) of tetracycline for 24 hours. The blot was hybridised with an ARL1-specific probe, and with ß-tubulin to monitor equal sample loading. (C) Nuclei and kinetoplasts were counted in parasitic line Bp2T7/ARL1 in the absence (light shading) and presence (dark shading) of tetracycline, over a 48 hour time course. Any configurations other than K1N1, K2N1 and K2N2 were classified as abnormal. 250 parasites were counted per experimental group. (D) Representative images of Bp2T7/ARL1 parasites with abnormal nucleus/kinetoplast configurations after induction with tetracycline for 36 hours. DAPI-stained cells are shown (a,b) with corresponding phase contrast images (a',b'). (E) Growth of the procyclic transfected line Pp2T7/ARL1 in the absence (open symbols) and presence (filled symbols) of tetracycline over a 5 day time course. Bar, 10 µm.

View larger version (93K): [in a new window]   Fig. 4. Electron micrographs of BSF line Bp2T7/ARL1. (A,B) Cells grown in the presence of tetracycline for 36 hours. F, flagellum; FP, flagellar pocket; GA, Golgi apparatus; MVB, multi-vesicular body; N, nucleus. The Golgi apparatus from cells grown in the absence (C) and presence (D-F) of tetracycline. Image D is an enlarged view (x4.5) of the boxed area in B. Bar, 1 µm (A,B); 0.2 µm (C-F).

View larger version (54K): [in a new window]   Fig. 5. Immunofluorescence of BSF line Bp2T7/ARL1 grown in the absence (-Tet) and presence (+Tet) of tetracycline for 24 and 36 hours. All cells are co-stained with DAPI (shown in blue). (A) ER-localized BiP staining (green). (B) Lysosomal marker, p67 (red). (C) Heavy chain of T. brucei clathrin (green). Bar, 10 µm.

View larger version (49K): [in a new window]   Fig. 6. Effects of ARL1 depletion on endocytosis. Receptor-mediated endocytosis was analysed by monitoring the uptake of FITC-labelled ConA in BSF line Bp2T7/ARL1 grown in the absence (-Tet) and presence (+Tet) of tetracycline for 24 and 36 hours. All cells are co-stained with DAPI (blue). (A) In cells incubated for 30 minutes at 12°C, ConA (green) is present in the endosomes. (B) In cells incubated for 30 minutes at 37°C, all ConA has reached the lysosome, except in parasites induced for 36 hours, which still contain a significant quantity of ConA in the endosomes. (C) Colocalisation of FITC-ConA (green) with the lysosomal marker p67 (red) in cells as in B. Bar, 10 µm.

View larger version (34K): [in a new window]   Fig. 7. Effects of ARL1 depletion on exocytosis and Golgi function. (A) VSG exocytosis assay in BSF line Bp2T7/ARL1 grown in the absence (No Tet) and presence (+Tet) of tetracycline for 24 hours. Parasites were labelled by pulse-chase using [35S]-labelled methionine and cysteine over a time course of 40 minutes and VSG isolated from cell lysates using ConA sepharose. Radiolabelled products were separated by SDS-PAGE and detected by autoradiography. Numbers represent length of pulse-chase (minutes). Insoluble pellet fractions (P) represent newly synthesized membrane-bound VSG in the endomembrane system, whereas soluble fractions (S) represent newly synthesized VSG delivered to the plasma membrane, where it is susceptible to the action of endogenous GPI-phospholipase C. The data shown represent one of three independent experiments. (B) Data from three assays as described in (A) were quantified by densitometry and mean values (±s.d.) are shown here as % VSG released on to the plasma membrane in BSF line Bp2T7/ARL1 grown in the absence (open squares) and presence (filled squares) of tetracycline for 24 hours. 0% is the ratio obtained at time 0 and 100% is the point at which all VSG is in the soluble fraction. (C,D) Immunoprecipitation of p67, following radioactive labelling of parasites by pulse chase with [35S]methionine and [35S]cysteine over a 4-hour time course. BSF line Bp2T7/ARL1 cells were grown in the absence (C) and presence (D) of tetracycline for 36 hours. Proteins were separated by SDS-PAGE and radiolabelled products detected by autoradiography. P67 is synthesized as a 100 kDa protein, which is modified by N-glycans to form a 150 kDa molecule, clearly visible in both samples by 30 minutes. After delivery to the lysosome, the protein is proteolytically cleaved into fragments of 75, 42, 32 and 28 kDa, as observed after 1 hour. Note that the lower fragment is not seen in this figure and that there is an unidentified band in samples from later time points with a molecular weight of approximately 55 kDa. (E,F) p67 immunoprecipitation results were quantified by densitometry in cells grown in the absence (light shading) and presence (dark shading) of tetracycline for 36 hours. (E) The percentage of total [35S]p67 present as the N-glycan modified glycoform (gp150) during the 4 hour pulse-chase time course. (F) Percentage of total [35S]p67 present as proteolytically cleaved fragments (gp75, 42 and 32 glycoforms) in the lysosome. Data shown represent one of two independent experiments.