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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 LdpGRIP-2.Overlay of fixed L. amazonensis promastigotes expressing in red mRed-LdpGRIP-2 and in green LdARL-1-GFP (A), LdARL-1/Q74L-GFP (GTP) (B), LdARL-1/G2A-GFP (unMyr) (C), LdARL-1/T51N-GFP (GDP) (D) and LdARL-1/T34N-GFP (empty) (E). Overlay of red and green is yellow.
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pone-0001620-g010: LdARL-1 and LdpGRIP-2.Overlay of fixed L. amazonensis promastigotes expressing in red mRed-LdpGRIP-2 and in green LdARL-1-GFP (A), LdARL-1/Q74L-GFP (GTP) (B), LdARL-1/G2A-GFP (unMyr) (C), LdARL-1/T51N-GFP (GDP) (D) and LdARL-1/T34N-GFP (empty) (E). Overlay of red and green is yellow.

Mentions: Fluorescence microscopy analyses revealed that mRed-LdpGRIP-2 co-localised to the TGN with LdARL-1-GFP (Fig 10A), LdARL-1/Q74L-GFP (Fig 10B), and LdARL-1/T51N-GFP (Fig 10D). Interestingly, LdARL-1/G2A-GFP, which remained cytoplasmic (cf Fig 4B, 6C), did not interfere with mRed-LdpGRIP-2 localisation (Fig 10C). Even more interestingly, LdARL-1/T34N-GFP inhibited the TGN addressing of mRed-LdpGRIP-2, as both proteins colocalised within the cytoplasm (Fig 10E). This observation is consistent with the TGN disorganisation observed in mammals by the equivalent mutant ARL-1/T31N overexpression [18] and probably explains the interruption of intracellular traffic from the TGN, since the vesicle tethers are no longer recruited to their location.


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 LdpGRIP-2.Overlay of fixed L. amazonensis promastigotes expressing in red mRed-LdpGRIP-2 and in green LdARL-1-GFP (A), LdARL-1/Q74L-GFP (GTP) (B), LdARL-1/G2A-GFP (unMyr) (C), LdARL-1/T51N-GFP (GDP) (D) and LdARL-1/T34N-GFP (empty) (E). Overlay of red and green is yellow.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001620-g010: LdARL-1 and LdpGRIP-2.Overlay of fixed L. amazonensis promastigotes expressing in red mRed-LdpGRIP-2 and in green LdARL-1-GFP (A), LdARL-1/Q74L-GFP (GTP) (B), LdARL-1/G2A-GFP (unMyr) (C), LdARL-1/T51N-GFP (GDP) (D) and LdARL-1/T34N-GFP (empty) (E). Overlay of red and green is yellow.
Mentions: Fluorescence microscopy analyses revealed that mRed-LdpGRIP-2 co-localised to the TGN with LdARL-1-GFP (Fig 10A), LdARL-1/Q74L-GFP (Fig 10B), and LdARL-1/T51N-GFP (Fig 10D). Interestingly, LdARL-1/G2A-GFP, which remained cytoplasmic (cf Fig 4B, 6C), did not interfere with mRed-LdpGRIP-2 localisation (Fig 10C). Even more interestingly, LdARL-1/T34N-GFP inhibited the TGN addressing of mRed-LdpGRIP-2, as both proteins colocalised within the cytoplasm (Fig 10E). This observation is consistent with the TGN disorganisation observed in mammals by the equivalent mutant ARL-1/T31N overexpression [18] and probably explains the interruption of intracellular traffic from the TGN, since the vesicle tethers are no longer recruited to their location.

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