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First published online 5 February 2008
doi: 10.1242/jcs.022574

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The AP3 adaptor is involved in the transport of membrane proteins to acidocalcisomes of Leishmania

Sébastien Besteiro^*, Daniela Tonn, Laurence Tetley, Graham H. Coombs and Jeremy C. Mottram

Wellcome Centre for Molecular Parasitology and Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 120 University Place, Glasgow, G12 8TA, UK

View larger version (39K): [in this window] [in a new window]   Fig. 1. Targeted replacement of the AP3 gene. (A, from left to right) Schematic representation of the AP3 locus and the constructs used for gene replacement. Genes are shown as arrows, intergenic and flanking DNA sequences as boxes. The restriction sites and the locations of the probes used for the Southern blot analyses, as well as the expected sizes of the labelled fragments, are displayed. HYG, hygromycin-resistance gene; BLE, phleomycin-resistance gene; DHFR, dihydrofolate reductase gene. Southern blot analyses of L. major wild-type (WT), AP3 heterozygote (ap3/AP3) and AP3-null mutant (ap3) with probes specific for HYG and AP3. (B) RT-PCR analysis of specific mRNA expression in promastigotes of the cell lines described in A and the additional AP3 re-expressing cell line (ap3::PRRNA AP3). Re-expression of AP3 is from the ribosomal DNA locus and contains a different 3'UTR from the wild type gene, so the AP3 mRNA is a different size. +RT and –RT, initial reaction with and without reverse transcriptase, respectively. VPS4-specific primers were used as a positive control.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Normal lysosome function in the ap3 mutant. (A) Gelatin-SDS-PAGE analysis to detect the activities of mature lysosomal cysteine peptidase CPB (arrowhead) and metallopeptidases (asterisk). (B) Metabolic labelling and immunoprecipitation analysis assessing the maturation of CPB in the wild-type and ap3 cell lines. The mature CPB form is indicated by an arrowhead and the precursor forms by asterisks. Molecular masses are indicated on the right. (C) Labelling of the MVT-lysosome (arrowheads) in L. major ap3 and wild-type promastigotes with GFP-tagged syntaxins LmjF19.0120 and LmjF29.0070 (green signal) and endocytic tracer FM4-64 (red signal). n, nucleus; k, kinetoplast; scale bars, 10 µm. Corresponding differential interference contrast (DIC) images are shown as well.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Acidocalcisome defects in ap3 promastigotes. (A, top panels) Lysotracker labelling of acidic compartments in L. major promastigotes. The two types of signals observed for the ap3 cell line, including the MVT-lysosome-like tubular labelling, are displayed in the four middle panels. (A, bottom panels) Acridine-Orange-labelling of acidic compartments in L. major promastigotes. (B) Measurements of Neutral Red change in absorbance at 540 nm due to acidic environments of L. major promastigotes. Data represent the means ± standard deviations (s.d.) from four independent experiments. (C) Visualisation of polyP-enriched vesicles (yellow) with DAPI in L. major promastigotes. n, nucleus; k, kinetoplast. All scale bars, 10 µm. Corresponding DIC images are shown as well.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Acidocalcisomal membrane V-H+-PPase is downregulated in ap3 promastigotes. (A) Western blot analysis of whole-cell lysates from L. major promastigotes with the anti-V-H+-PPase antibody (VP1). Elongation factor 1 (EF1-) was used as a loading control. Molecular sizes are indicated on the left. (B) Immunofluorescence analysis of L. major promastigotes using the anti-V-H+-PPase antibody. (C) Localisation in WT and ap3 L. major promastigotes, of the V-H+-PPase with GFP fused at its C-terminal end. All scale bars, 10 µm; n, nucleus; k, kinetoplast. Corresponding DIC images are shown as well.

View larger version (18K): [in this window] [in a new window]   Fig. 5. ap3 promastigotes are defective in PPi- and ATP-dependent proton transport. (A,B) Proton-pumping activities visualised in permeabilised promastigotes loaded with Acridine Orange, upon addition of (A) 500 µM NaPPi or (B) 1 mM ATP. 20 mM of NH4Cl was added to reverse the proton gradient when indicated. The horizontal scales represent time and the vertical scales the absorbance variations of Acridine Orange at 530 nm and 493 nm following pH changes. Data shown are representative of three experiments performed with independent cultures.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Acidocalcisomes from ap3 promastigotes contain a soluble pyrophosphatase, but are devoid of electron-dense material. (A) Western blot analysis of whole-cell lysates from L. major promastigotes with the anti-VSP1 antibody. EF1- was used as a loading control. Molecular masses are indicated on the left. (B) Immunofluorescence analysis of L. major promastigotes using anti-VSP1 antibody. The corresponding DIC images are shown. (C, left panels) electron spectroscopic whole cell imaging of WT, ap3 and ap3::PRRNA AP3 promastigotes showing presence or absence of electron-dense polyphosphate in acidocalcisomes (arrowheads). (Right panels) Corresponding transmission electron microscopy of sections of these cell types. Acidocalcisomes are indicated with arrowheads. Nuclei (n) and glycosomes (arrows) are indicated as other organelles for reference; k, kinetoplast. Scale bars, 10 µm (B) and 2.0 µm (C).

View larger version (35K): [in this window] [in a new window]   Fig. 7. AP3 is a virulence factor. (A) In vitro growth of promastigotes. Data shown represent the means ± s.d. from three independent series of measurements. (B) Western blot analysis with metacyclic-specific anti-HASPB antibody. EF1- was used as a loading control. Molecular masses are indicated on the right. (C) In-vitro-macrophage-infectivity assay. Stationary-phase parasites were used to infect explanted mouse peritoneal macrophages at a 3:1 ratio for 4 hours. The proportion of infected macrophages (left) and the number of parasites per infected macrophage (right) were determined after 5 days of incubation. Data shown represent are the means ± s.d. from triplicate infections. *, results for ap3 cells that differ significantly from those for the other two cell lines (t-test, P<0.05). (D) Infection of CD1 mice with wild-type and transgenic stationary-phase L. major promastigotes. Mice were challenged with 5x105 parasites in the left hind footpad. The swelling caused by the respective cell lines was recorded. Data shown represent mean lesion depth ± s.d. from groups of six mice.