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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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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).
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pone-0001620-g004: 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).

Mentions: Indirect immunofluorescence microscopy observations with the anti-LdARL-1 antiserum revealed a spot in the vicinity of the kinetoplast and the flagellar pocket (Fig 3A), strongly reminiscent of the TGN localization reported for LdARF-1-GFP [10]. Promastigotes expressing LdARL-1-GFP showed the same localisation (Fig 3B, 4A); the green GFP signal was adjacent to the red one of mRed-LdLpg-2 signal, a Golgi cisternae marker [40] (Fig 3B) and co-localised with LdARF-1-mRed [10] (Fig 3C). Electron microscopy confirmed the LdARL-1 TGN localisation (Fig 3D). In the ARL-1 sequence of L. donovani, L. amazonensis, L. major and T. brucei, the Golgi localisation signal MXXE [41] present in HsARL-1, ScARL-1 and HsARF-1 (Fig 1) is replaced by LXXE, likely functional according to our IF (immunofluorescence) results.


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 the non-myristoylated LdARL-1.Fixed L. amazonensis promastigotes transformed with: A, pNUS-LdARL-1-GFPcH (green); B, pNUS-LdARL-1/G2A-GFPcH (green).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001620-g004: 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).
Mentions: Indirect immunofluorescence microscopy observations with the anti-LdARL-1 antiserum revealed a spot in the vicinity of the kinetoplast and the flagellar pocket (Fig 3A), strongly reminiscent of the TGN localization reported for LdARF-1-GFP [10]. Promastigotes expressing LdARL-1-GFP showed the same localisation (Fig 3B, 4A); the green GFP signal was adjacent to the red one of mRed-LdLpg-2 signal, a Golgi cisternae marker [40] (Fig 3B) and co-localised with LdARF-1-mRed [10] (Fig 3C). Electron microscopy confirmed the LdARL-1 TGN localisation (Fig 3D). In the ARL-1 sequence of L. donovani, L. amazonensis, L. major and T. brucei, the Golgi localisation signal MXXE [41] present in HsARL-1, ScARL-1 and HsARF-1 (Fig 1) is replaced by LXXE, likely functional according to our IF (immunofluorescence) results.

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