Figure 1.
Sequence analysis of ARL-1/ARF-1 of various species.
Consensus motives for the GDP-GTP binding site are boxed in black. Mutated and amino acids of interest are boxed in grey. The sequence of the C-terminal peptide synthesised for the production of the rabbit immune serum is underlined. Databanks accession numbers: LdARL-1, Genbank AF187855 and AAF29899; LaARL-1, GenBank XXX; LmARL-1, GeneDB LmjF17.0070 (http://www.genedb.org/); TbARL-1, Genbank AAX70381, GeneDB Tb927.7.6230, previously Tb07.2F2.550; HsARL-1, Swissprot P40616; ScARL-1: Genbank S46035; HsARF-1: Swissprot P32889; HsARF-6: Genbank NP_001654.
Figure 2.
Expression of LdARL-1 and mutants in L. amazonensis.
Extracts of 3.106 exponentially growing L. amazonensis promastigotes (about 3 µg of proteins) were submitted to western blot analysis using the rabbit anti-LdARL-1 C-terminus immune serum (1∶5000 dilution); color was developed with an anti-rabbit IgG peroxidase conjugate (1∶10000 dilution) and an ECL revelation kit (Amersham). Internal standard: 37 kDa LACK antigen. Left, negative control without anti-LdARL-1 antiserum. Additional bands are revealed when the GFP-fused proteins are expressed, probably due to partial degradation when the cells were lysed, in spite of the presence of antiproteases (see Methods).
Figure 3.
LdARL-1 subcellular localisation in L. amazonensis promastigotes.
A. IF on fixed cells: A1, phase contrast; A2, co-labelling with DAPI (blue) and FITC-anti rabbit-IgG plus anti-LdARL-1 rabbit immune serum (green). B. IF on fixed cells co-transformed with pNUS-LdARL-1-GFPcH (green) and pNUS-mREDnD-LdLPG-2 (red) plus DAPI staining (blue). C. IF on fixed cells co-transformed with pNUS-LdARL-1-GFPcH (C1, green) plus pNUS-LdARF-1-mRedcD (C2, red), overlay (C3). D. EM of cells transformed with pNUS-LdARL-1-GFP and incubated with anti-LdARL-1 immune serum. C1 and C2 are sections of two different cells. Black dots (10 nm gold particles) show the LdARL-1 immunoreactive spots. N: nucleus; G: Golgi apparatus; K: kinetoplast; F: flagellum; FP: flagellar pocket.
Figure 4.
Localisation of the non-myristoylated LdARL-1.
Fixed L. amazonensis promastigotes transformed with: A, pNUS-LdARL-1-GFPcH (green); B, pNUS-LdARL-1/G2A-GFPcH (green).
Figure 5.
Localisation of LdARL-1-GFP mutants (GTP-, GDP-bound and empty forms).
Fixed L. amazonensis promastigotes expressing in red LdARF-1-mRed (5B, 5E, 5H) and in green LdARL-1/Q74L-GFP (GTP) (5A), LdARL-1/T51N-GFP (GDP) (5D), LdARL-1/T34N-GFP (empty) (5H); overlays are in 5C, 5F and 5I. fp, flagellar pocket.
Figure 6.
LdARL-1 and the flagellar pocket.
Fixed L. amazonensis promastigotes with plasma membrane labelled in red with Concanavalin A-Texas Red. A, cell expressing in green LdARL-1-GFP. B-C, cell expressing in green LdARL-1/T51N-GFP (GDP); D-F, cell expressing in green LdARL-1/T34N-GFP (empty). The yellow signal is an overlay of green and red signals.
Figure 7.
LdARL-1 and FM4-64 internalisation.
L. amazonensis promastigotes expressing LdARL-1-GFP (A–D, green) or LdARL-1/T34N-GFP (empty) (E–H, green) were incubated at 24°C for 15 min with 2 µg/ml FM4-64FX (red), washed and further incubated for 0 min (A, E), 15 min (B, F), 60 min (C, G) and 120 min (D, H) before fixation; fp, flagellar pocket; MVT, Lysosome/Multivesicular Tubule. Colocalization of red and green results in a yellow signal.
Figure 8.
LdARL-1 and Golgi to Lysosome/MVT traffic.
BA125 L. amazonensis promastigotes expressing in red the mRed-DPMS and in green LdARL-1-GFP (A), LdARL-1/G2A-GFP (unMyr) (B), LdARL-1/Q74L-GFP (GTP) (C), LdARL-1/T51N-GFP (GDP) (D) or LdARL-1/T34N-GFP (empty) (E).
Figure 9.
A. Fixed L. amazonensis promastigotes expressing in green LdARL-1-GFP (1), LdARL-1/Q74L-GFP (GTP) (2), LdARL-1/T51N-GFP (GDP) (3), LdARL-1/G2A-GFP (unMyr) (4) or LdARL-1/T34N-GFP (empty) (5) and stained in red with the rabbit anti-TbVP1 (acidocalcisomes marker) immune serum plus anti-rabbit IgG-Texas-Red conjugate. B. Live L. amazonensis promastigotes expressing in green LdARL-1-GFP (1-2), LdARL-1/T34N-GFP (empty) (3-4) or LdARL-1/T51N-GFP (GDP) (5-6) and stained with DAPI: in Blue, nucleus and kinetoplast, in yellow: polyphosphates of acidocalcisomes [50].
Figure 10.
Overlay of fixed L. amazonensis promastigotes expressing in red mRed-LdpGRIP-2 and in green LdARL-1-GFP (A), LdARL-1/Q74L-GFP (GTP) (B), LdARL-1/G2A-GFP (unMyr) (C), LdARL-1/T51N-GFP (GDP) (D) and LdARL-1/T34N-GFP (empty) (E). Overlay of red and green is yellow.