Limits...
The leishmania ARL-1 and Golgi traffic.

Sahin A, Espiau B, Tetaud E, Cuvillier A, Lartigue L, Ambit A, Robinson DR, Merlin G - PLoS ONE (2008)

Bottom Line: This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form.The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N.Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Microbiologie Cellulaire et Moléculaire et Pathogénicité, UMR CNRS 5234, Université Bordeaux 2, Bordeaux, France.

ABSTRACT
We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2237903&req=5

pone-0001620-g007: 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.

Mentions: Endocytosis was assessed by FM4-64 pulse-chase experiments (Fig 7). In Leishmania, this dye is readily internalised and targeted through endosomes to the final digestive compartment termed Lysosome/Multivesicular Tubule (MVT) [46]. In cells expressing LdARL-1-GFP (Fig 7A–D), LdARL-1/Q74L-GFP (not shown) or LdARL-1/T51N-GFP (not shown), FM4-64 endocytosis occurred as expected, with a progressive and massive FM4-64 labelling from the flagellar pocket to the MVT from 0 to 120 minutes. On the contrary, cells expressing LdARL-1/T34N-GFP showed endocytosis defect (Fig 7E–H); the FM4-64 uptake was blocked, the labelling, remaining in the area of the flagellar pocket up to 60 minutes (Fig 7E–G, arrows), was only seen progressing to the MVT at later times (120 min) and in much smaller amounts (Fig 7H, arrow); as comparison, the cell a in Fig 7F, which expresses LdARL-1/T34N-GFP, and the cell b which does not stresses the difference in FM4-64 internalization. This endocytosis defect was not due to clathrin depletion, as revealed by immunofluorescence with an anti-TbClathrin Heavy Chain immune serum (not shown).


The leishmania ARL-1 and Golgi traffic.

Sahin A, Espiau B, Tetaud E, Cuvillier A, Lartigue L, Ambit A, Robinson DR, Merlin G - PLoS ONE (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2237903&req=5

pone-0001620-g007: 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.
Mentions: Endocytosis was assessed by FM4-64 pulse-chase experiments (Fig 7). In Leishmania, this dye is readily internalised and targeted through endosomes to the final digestive compartment termed Lysosome/Multivesicular Tubule (MVT) [46]. In cells expressing LdARL-1-GFP (Fig 7A–D), LdARL-1/Q74L-GFP (not shown) or LdARL-1/T51N-GFP (not shown), FM4-64 endocytosis occurred as expected, with a progressive and massive FM4-64 labelling from the flagellar pocket to the MVT from 0 to 120 minutes. On the contrary, cells expressing LdARL-1/T34N-GFP showed endocytosis defect (Fig 7E–H); the FM4-64 uptake was blocked, the labelling, remaining in the area of the flagellar pocket up to 60 minutes (Fig 7E–G, arrows), was only seen progressing to the MVT at later times (120 min) and in much smaller amounts (Fig 7H, arrow); as comparison, the cell a in Fig 7F, which expresses LdARL-1/T34N-GFP, and the cell b which does not stresses the difference in FM4-64 internalization. This endocytosis defect was not due to clathrin depletion, as revealed by immunofluorescence with an anti-TbClathrin Heavy Chain immune serum (not shown).

Bottom Line: This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form.The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N.Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Microbiologie Cellulaire et Moléculaire et Pathogénicité, UMR CNRS 5234, Université Bordeaux 2, Bordeaux, France.

ABSTRACT
We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

Show MeSH
Related in: MedlinePlus