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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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LdARL-1 and acidocalcisomes.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].
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pone-0001620-g009: LdARL-1 and acidocalcisomes.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].

Mentions: The VP1 orthologue exists in L. amazonensis (LaVP1) and can be detected by immunofluorescence, using the anti-TbVP1 immune serum (N. Bakalara, personal communication), within large round-shaped organelles, distributed thoughout the promastigote body. Using the TbVP1 antibody, this characteristic pattern of acidocalcisomes was observed in L. amazonensis promastigotes expressing LdARL-1-GFP (Fig 9A-1), LdARL-1/Q74L-GFP (Fig 9A-2), LdARL-1/T51N-GFP (Fig 9A-3) and the delocalised/inert LdARL-1/G2A-GFP (Fig 9A-4). Strikingly, in cells expressing LdARL-1/T34N-GFP, TbVP1 was undetectable (Fig 9A-5). However, DAPI staining of live cells, which allows visualisation of their acidocalcisomes by revealing their polyphosphate content, showed that all cells expressing LdARL-1-GFP (Fig 9B-1), LdARL-1/T34N-GFP (Fig 9B-2) and LdARL-1/T51N-GFP (Fig 9B-3) contained polyphosphate/acidocalcisomes; this ruled out the possible absence of these organelles in the LdARL-1/T34N-GFP expressing cells. Therefore, in L. amazonensis, expression of LdARL-1/T34N-GFP induced a misdirection, possibly exocytosis, of the membrane-bound proton pyrophosphatase.


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 acidocalcisomes.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].
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Related In: Results  -  Collection

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pone-0001620-g009: LdARL-1 and acidocalcisomes.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].
Mentions: The VP1 orthologue exists in L. amazonensis (LaVP1) and can be detected by immunofluorescence, using the anti-TbVP1 immune serum (N. Bakalara, personal communication), within large round-shaped organelles, distributed thoughout the promastigote body. Using the TbVP1 antibody, this characteristic pattern of acidocalcisomes was observed in L. amazonensis promastigotes expressing LdARL-1-GFP (Fig 9A-1), LdARL-1/Q74L-GFP (Fig 9A-2), LdARL-1/T51N-GFP (Fig 9A-3) and the delocalised/inert LdARL-1/G2A-GFP (Fig 9A-4). Strikingly, in cells expressing LdARL-1/T34N-GFP, TbVP1 was undetectable (Fig 9A-5). However, DAPI staining of live cells, which allows visualisation of their acidocalcisomes by revealing their polyphosphate content, showed that all cells expressing LdARL-1-GFP (Fig 9B-1), LdARL-1/T34N-GFP (Fig 9B-2) and LdARL-1/T51N-GFP (Fig 9B-3) contained polyphosphate/acidocalcisomes; this ruled out the possible absence of these organelles in the LdARL-1/T34N-GFP expressing cells. Therefore, in L. amazonensis, expression of LdARL-1/T34N-GFP induced a misdirection, possibly exocytosis, of the membrane-bound proton pyrophosphatase.

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