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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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Sequence analysis of ARL-1/ARF-1 of various species.Consensus motives for the GDP-GTP binding site are boxed in black. Mutated and amino acids of interest are boxed in grey. The sequence of the C-terminal peptide synthesised for the production of the rabbit immune serum is underlined. Databanks accession numbers: LdARL-1, Genbank AF187855 and AAF29899; LaARL-1, GenBank XXX; LmARL-1, GeneDB LmjF17.0070 (http://www.genedb.org/); TbARL-1, Genbank AAX70381, GeneDB Tb927.7.6230, previously Tb07.2F2.550; HsARL-1, Swissprot P40616; ScARL-1: Genbank S46035; HsARF-1: Swissprot P32889; HsARF-6: Genbank NP_001654.
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pone-0001620-g001: Sequence analysis of ARL-1/ARF-1 of various species.Consensus motives for the GDP-GTP binding site are boxed in black. Mutated and amino acids of interest are boxed in grey. The sequence of the C-terminal peptide synthesised for the production of the rabbit immune serum is underlined. Databanks accession numbers: LdARL-1, Genbank AF187855 and AAF29899; LaARL-1, GenBank XXX; LmARL-1, GeneDB LmjF17.0070 (http://www.genedb.org/); TbARL-1, Genbank AAX70381, GeneDB Tb927.7.6230, previously Tb07.2F2.550; HsARL-1, Swissprot P40616; ScARL-1: Genbank S46035; HsARF-1: Swissprot P32889; HsARF-6: Genbank NP_001654.

Mentions: The predicted ARL-1 protein, which contains 187 amino acids (20 833 Da, pI 5.24), displays 55% identity with the human HsARL-1, 52% with yeast ScARL-1 and 50% with human HsARF-1 (Fig 1). As for all ARFs/ARLs, the Glycine at position 2 is a potential myristoylation site. The first three consensus motifs of the GDP/GTP binding site (GxxxxGKT, DxxG, and NxxD) [36] are present (Fig 1) while the fourth one (C/SA/xx) is less conserved as often observed for ARFs/ARLs. The ability of the recombinant LdARL-1 to bind GTPγS has been described elsewhere [37].


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)

Sequence analysis of ARL-1/ARF-1 of various species.Consensus motives for the GDP-GTP binding site are boxed in black. Mutated and amino acids of interest are boxed in grey. The sequence of the C-terminal peptide synthesised for the production of the rabbit immune serum is underlined. Databanks accession numbers: LdARL-1, Genbank AF187855 and AAF29899; LaARL-1, GenBank XXX; LmARL-1, GeneDB LmjF17.0070 (http://www.genedb.org/); TbARL-1, Genbank AAX70381, GeneDB Tb927.7.6230, previously Tb07.2F2.550; HsARL-1, Swissprot P40616; ScARL-1: Genbank S46035; HsARF-1: Swissprot P32889; HsARF-6: Genbank NP_001654.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001620-g001: Sequence analysis of ARL-1/ARF-1 of various species.Consensus motives for the GDP-GTP binding site are boxed in black. Mutated and amino acids of interest are boxed in grey. The sequence of the C-terminal peptide synthesised for the production of the rabbit immune serum is underlined. Databanks accession numbers: LdARL-1, Genbank AF187855 and AAF29899; LaARL-1, GenBank XXX; LmARL-1, GeneDB LmjF17.0070 (http://www.genedb.org/); TbARL-1, Genbank AAX70381, GeneDB Tb927.7.6230, previously Tb07.2F2.550; HsARL-1, Swissprot P40616; ScARL-1: Genbank S46035; HsARF-1: Swissprot P32889; HsARF-6: Genbank NP_001654.
Mentions: The predicted ARL-1 protein, which contains 187 amino acids (20 833 Da, pI 5.24), displays 55% identity with the human HsARL-1, 52% with yeast ScARL-1 and 50% with human HsARF-1 (Fig 1). As for all ARFs/ARLs, the Glycine at position 2 is a potential myristoylation site. The first three consensus motifs of the GDP/GTP binding site (GxxxxGKT, DxxG, and NxxD) [36] are present (Fig 1) while the fourth one (C/SA/xx) is less conserved as often observed for ARFs/ARLs. The ability of the recombinant LdARL-1 to bind GTPγS has been described elsewhere [37].

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