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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

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.
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pone-0001620-g005: 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.

Mentions: The growth rate of the cell lines expressing LdARL-1/Q74L or LdARL-1/T51N were comparable (Fig S2). Both proteins localised to the TGN (Fig 5A and D) and colocalised with the TGN marker LdARF-1-mRed (Fig 5B/5C and 5E/5F, respectively). Remarkably, while expressed at similar levels as LdARL-1/Q74L-GFP (Fig 2), the double-mutant LdARL-1/G2AQ74L-GFP remained cytoplasmic (not shown), as did LdARL-1/G2A-GFP, thus confirming that myristoylation is mandatory for the TGN localisation.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001620-g005: 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.
Mentions: The growth rate of the cell lines expressing LdARL-1/Q74L or LdARL-1/T51N were comparable (Fig S2). Both proteins localised to the TGN (Fig 5A and D) and colocalised with the TGN marker LdARF-1-mRed (Fig 5B/5C and 5E/5F, respectively). Remarkably, while expressed at similar levels as LdARL-1/Q74L-GFP (Fig 2), the double-mutant LdARL-1/G2AQ74L-GFP remained cytoplasmic (not shown), as did LdARL-1/G2A-GFP, thus confirming that myristoylation is mandatory for the TGN localisation.

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