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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.

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Related in: MedlinePlus

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.
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pone-0001620-g006: 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.

Mentions: The trypanosomatids are polarised cells in which endo- and exocytosis occur solely in a peculiar structure, the flagellar pocket. Upon exogenous expression of LdARL-1/T34N-GFP (empty), the flagellar pocket of the transgenic L. amazonensis cells appeared inflated, forming a gap at the anterior part of the cells (Fig 5G, fp); this was better resolved by labelling the pellicular and pocket membranes with Texas-Red-Concanavalin A, as seen for the cell pictured on Fig 6D–G and comparing to a cell expressing native LdARL-1-GFP (Fig 6A) or LdARL-1/T51N-GFP (GDP) (Fig 6B–C). This flagellar pocket inflation was similar to the observation made when endocytosis was inhibited by RNAi ablation of clathrin heavy chain in T. brucei [45], but could also result from enhanced exocytosis.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001620-g006: 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.
Mentions: The trypanosomatids are polarised cells in which endo- and exocytosis occur solely in a peculiar structure, the flagellar pocket. Upon exogenous expression of LdARL-1/T34N-GFP (empty), the flagellar pocket of the transgenic L. amazonensis cells appeared inflated, forming a gap at the anterior part of the cells (Fig 5G, fp); this was better resolved by labelling the pellicular and pocket membranes with Texas-Red-Concanavalin A, as seen for the cell pictured on Fig 6D–G and comparing to a cell expressing native LdARL-1-GFP (Fig 6A) or LdARL-1/T51N-GFP (GDP) (Fig 6B–C). This flagellar pocket inflation was similar to the observation made when endocytosis was inhibited by RNAi ablation of clathrin heavy chain in T. brucei [45], but could also result from enhanced exocytosis.

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