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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

Expression of LdARL-1 and mutants in L. amazonensis.Extracts of 3.106 exponentially growing L. amazonensis promastigotes (about 3 µg of proteins) were submitted to western blot analysis using the rabbit anti-LdARL-1 C-terminus immune serum (1∶5000 dilution); color was developed with an anti-rabbit IgG peroxidase conjugate (1∶10000 dilution) and an ECL revelation kit (Amersham). Internal standard: 37 kDa LACK antigen. Left, negative control without anti-LdARL-1 antiserum. Additional bands are revealed when the GFP-fused proteins are expressed, probably due to partial degradation when the cells were lysed, in spite of the presence of antiproteases (see Methods).
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pone-0001620-g002: Expression of LdARL-1 and mutants in L. amazonensis.Extracts of 3.106 exponentially growing L. amazonensis promastigotes (about 3 µg of proteins) were submitted to western blot analysis using the rabbit anti-LdARL-1 C-terminus immune serum (1∶5000 dilution); color was developed with an anti-rabbit IgG peroxidase conjugate (1∶10000 dilution) and an ECL revelation kit (Amersham). Internal standard: 37 kDa LACK antigen. Left, negative control without anti-LdARL-1 antiserum. Additional bands are revealed when the GFP-fused proteins are expressed, probably due to partial degradation when the cells were lysed, in spite of the presence of antiproteases (see Methods).

Mentions: A rabbit immune serum raised against the last 15 aa of LdARL-1 (underlined in Fig 1) allowed the detection of a unique 20 kDa band in promastigote and amastigote extracts of two strains of L. amazonensis, the BA125 strain (Fig. 2) and the LV79 strain (not shown). The expression of ARL-1 was similar in both forms of the parasite.


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)

Expression of LdARL-1 and mutants in L. amazonensis.Extracts of 3.106 exponentially growing L. amazonensis promastigotes (about 3 µg of proteins) were submitted to western blot analysis using the rabbit anti-LdARL-1 C-terminus immune serum (1∶5000 dilution); color was developed with an anti-rabbit IgG peroxidase conjugate (1∶10000 dilution) and an ECL revelation kit (Amersham). Internal standard: 37 kDa LACK antigen. Left, negative control without anti-LdARL-1 antiserum. Additional bands are revealed when the GFP-fused proteins are expressed, probably due to partial degradation when the cells were lysed, in spite of the presence of antiproteases (see Methods).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001620-g002: Expression of LdARL-1 and mutants in L. amazonensis.Extracts of 3.106 exponentially growing L. amazonensis promastigotes (about 3 µg of proteins) were submitted to western blot analysis using the rabbit anti-LdARL-1 C-terminus immune serum (1∶5000 dilution); color was developed with an anti-rabbit IgG peroxidase conjugate (1∶10000 dilution) and an ECL revelation kit (Amersham). Internal standard: 37 kDa LACK antigen. Left, negative control without anti-LdARL-1 antiserum. Additional bands are revealed when the GFP-fused proteins are expressed, probably due to partial degradation when the cells were lysed, in spite of the presence of antiproteases (see Methods).
Mentions: A rabbit immune serum raised against the last 15 aa of LdARL-1 (underlined in Fig 1) allowed the detection of a unique 20 kDa band in promastigote and amastigote extracts of two strains of L. amazonensis, the BA125 strain (Fig. 2) and the LV79 strain (not shown). The expression of ARL-1 was similar in both forms of the parasite.

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