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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 Golgi to Lysosome/MVT traffic.BA125 L. amazonensis promastigotes expressing in red the mRed-DPMS and in green LdARL-1-GFP (A), LdARL-1/G2A-GFP (unMyr) (B), LdARL-1/Q74L-GFP (GTP) (C), LdARL-1/T51N-GFP (GDP) (D) or LdARL-1/T34N-GFP (empty) (E).
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pone-0001620-g008: LdARL-1 and Golgi to Lysosome/MVT traffic.BA125 L. amazonensis promastigotes expressing in red the mRed-DPMS and in green LdARL-1-GFP (A), LdARL-1/G2A-GFP (unMyr) (B), LdARL-1/Q74L-GFP (GTP) (C), LdARL-1/T51N-GFP (GDP) (D) or LdARL-1/T34N-GFP (empty) (E).

Mentions: To further investigate the endocytosis deficiency observed in the LdARL-1/T34N-GFP-expressing cells, the dolichol-phosphate-mannose synthase (DPMS) was used as marker (Fig 8). In Leishmania, this membrane-anchored protein enzyme, essential for the biosynthesis of glycophosphatidylinositol anchors of membrane proteins, localises to the transitional Endoplasmic Reticulum [47]. However, GFP-DPMS chimeras are transported, via the Golgi apparatus and multivesicular bodies, to the lysosome/MVT [48]. A similar observation was made with an mRed-DPMS chimera in our cells expressing native LdARL-1-GFP (Fig 8A), LdARL-1/G2A-GFP (Fig 8B), LdARL-1/Q74L-GFP (Fig 8C) and LdARL-1/T51N-GFP (Fig 8D). In contrast, the mRed-DPMS chimera was undectectable in cells expressing the empty form LdARL-1/T34N-GFP (Fig 8E); as shown on Fig 8E, there is a clear mutual exclusion between the expression of LdARL-1/T34N-GFP (cell a) and mRed-DPMS detection (cell b). This suggests an alteration of the secretion pathway and/or a traffic misdirection.


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 Golgi to Lysosome/MVT traffic.BA125 L. amazonensis promastigotes expressing in red the mRed-DPMS and in green LdARL-1-GFP (A), LdARL-1/G2A-GFP (unMyr) (B), LdARL-1/Q74L-GFP (GTP) (C), LdARL-1/T51N-GFP (GDP) (D) or LdARL-1/T34N-GFP (empty) (E).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001620-g008: LdARL-1 and Golgi to Lysosome/MVT traffic.BA125 L. amazonensis promastigotes expressing in red the mRed-DPMS and in green LdARL-1-GFP (A), LdARL-1/G2A-GFP (unMyr) (B), LdARL-1/Q74L-GFP (GTP) (C), LdARL-1/T51N-GFP (GDP) (D) or LdARL-1/T34N-GFP (empty) (E).
Mentions: To further investigate the endocytosis deficiency observed in the LdARL-1/T34N-GFP-expressing cells, the dolichol-phosphate-mannose synthase (DPMS) was used as marker (Fig 8). In Leishmania, this membrane-anchored protein enzyme, essential for the biosynthesis of glycophosphatidylinositol anchors of membrane proteins, localises to the transitional Endoplasmic Reticulum [47]. However, GFP-DPMS chimeras are transported, via the Golgi apparatus and multivesicular bodies, to the lysosome/MVT [48]. A similar observation was made with an mRed-DPMS chimera in our cells expressing native LdARL-1-GFP (Fig 8A), LdARL-1/G2A-GFP (Fig 8B), LdARL-1/Q74L-GFP (Fig 8C) and LdARL-1/T51N-GFP (Fig 8D). In contrast, the mRed-DPMS chimera was undectectable in cells expressing the empty form LdARL-1/T34N-GFP (Fig 8E); as shown on Fig 8E, there is a clear mutual exclusion between the expression of LdARL-1/T34N-GFP (cell a) and mRed-DPMS detection (cell b). This suggests an alteration of the secretion pathway and/or a traffic misdirection.

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