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Two human ARFGAPs associated with COP-I-coated vesicles.

Frigerio G, Grimsey N, Dale M, Majoul I, Duden R - Traffic (2007)

Bottom Line: Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability.However, silencing all three ARFGAPs causes cell death.Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 2XY, United Kingdom.

ABSTRACT
ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

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ARFGAP domain and conserved motif of a representative subset of Glo3 proteins. A) The N-terminal ARFGAP domains of Glo3 and Gcs1 proteins from Saccharomyces cerevisiae, Schizosaccharomyces pombe and Homo sapiens were aligned using the clustal V algorithm in the MegAlign program (DNAStar package). Note the conserved cysteine residues highlighted in yellow and the overall high degree of sequence conservation in this domain. B). The Glo3 motif (highlighted in orange) consists of a repeat of 15 residues separated by 11–17 residues. It is a signature motif that allows unambiguous identification of Glo3 orthologues. Genbank accession numbers: cerevisiae: 6320969 (S. cerevisiae); Plasmodium 23478251 (P. yoelii); worm: 25153991 (Caenorhabditis elegans); fly: 24668642 (Drosophila melanogaster); plant A: 7487780, plant B: 18403775, plant C 15237500 (Arabidopsis thaliana); fish: assembled from several ESTs: 12148139, 12171549, 12158629 and 12265392 (Danio rerio); frog: 27695479 (Xenopus laevis) and human A: 21263420 and human B: 31543983 (H. sapiens).
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fig01: ARFGAP domain and conserved motif of a representative subset of Glo3 proteins. A) The N-terminal ARFGAP domains of Glo3 and Gcs1 proteins from Saccharomyces cerevisiae, Schizosaccharomyces pombe and Homo sapiens were aligned using the clustal V algorithm in the MegAlign program (DNAStar package). Note the conserved cysteine residues highlighted in yellow and the overall high degree of sequence conservation in this domain. B). The Glo3 motif (highlighted in orange) consists of a repeat of 15 residues separated by 11–17 residues. It is a signature motif that allows unambiguous identification of Glo3 orthologues. Genbank accession numbers: cerevisiae: 6320969 (S. cerevisiae); Plasmodium 23478251 (P. yoelii); worm: 25153991 (Caenorhabditis elegans); fly: 24668642 (Drosophila melanogaster); plant A: 7487780, plant B: 18403775, plant C 15237500 (Arabidopsis thaliana); fish: assembled from several ESTs: 12148139, 12171549, 12158629 and 12265392 (Danio rerio); frog: 27695479 (Xenopus laevis) and human A: 21263420 and human B: 31543983 (H. sapiens).

Mentions: Both Glo3p and Gcs1p share high sequence homology in their amino terminal catalytic zinc finger domain, which is known to engage ARF. Figure 1A shows an alignment of Glo3p, Gcs1p and their orthologues from S. pombe, as well as rat and human ARFGAP1, and the human ARFGAP2 and ARFGAP3. Note the presence of the four conserved cysteine residues characteristic for the zinc finger domain of ARFGAPs highlighted in yellow.


Two human ARFGAPs associated with COP-I-coated vesicles.

Frigerio G, Grimsey N, Dale M, Majoul I, Duden R - Traffic (2007)

ARFGAP domain and conserved motif of a representative subset of Glo3 proteins. A) The N-terminal ARFGAP domains of Glo3 and Gcs1 proteins from Saccharomyces cerevisiae, Schizosaccharomyces pombe and Homo sapiens were aligned using the clustal V algorithm in the MegAlign program (DNAStar package). Note the conserved cysteine residues highlighted in yellow and the overall high degree of sequence conservation in this domain. B). The Glo3 motif (highlighted in orange) consists of a repeat of 15 residues separated by 11–17 residues. It is a signature motif that allows unambiguous identification of Glo3 orthologues. Genbank accession numbers: cerevisiae: 6320969 (S. cerevisiae); Plasmodium 23478251 (P. yoelii); worm: 25153991 (Caenorhabditis elegans); fly: 24668642 (Drosophila melanogaster); plant A: 7487780, plant B: 18403775, plant C 15237500 (Arabidopsis thaliana); fish: assembled from several ESTs: 12148139, 12171549, 12158629 and 12265392 (Danio rerio); frog: 27695479 (Xenopus laevis) and human A: 21263420 and human B: 31543983 (H. sapiens).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171037&req=5

fig01: ARFGAP domain and conserved motif of a representative subset of Glo3 proteins. A) The N-terminal ARFGAP domains of Glo3 and Gcs1 proteins from Saccharomyces cerevisiae, Schizosaccharomyces pombe and Homo sapiens were aligned using the clustal V algorithm in the MegAlign program (DNAStar package). Note the conserved cysteine residues highlighted in yellow and the overall high degree of sequence conservation in this domain. B). The Glo3 motif (highlighted in orange) consists of a repeat of 15 residues separated by 11–17 residues. It is a signature motif that allows unambiguous identification of Glo3 orthologues. Genbank accession numbers: cerevisiae: 6320969 (S. cerevisiae); Plasmodium 23478251 (P. yoelii); worm: 25153991 (Caenorhabditis elegans); fly: 24668642 (Drosophila melanogaster); plant A: 7487780, plant B: 18403775, plant C 15237500 (Arabidopsis thaliana); fish: assembled from several ESTs: 12148139, 12171549, 12158629 and 12265392 (Danio rerio); frog: 27695479 (Xenopus laevis) and human A: 21263420 and human B: 31543983 (H. sapiens).
Mentions: Both Glo3p and Gcs1p share high sequence homology in their amino terminal catalytic zinc finger domain, which is known to engage ARF. Figure 1A shows an alignment of Glo3p, Gcs1p and their orthologues from S. pombe, as well as rat and human ARFGAP1, and the human ARFGAP2 and ARFGAP3. Note the presence of the four conserved cysteine residues characteristic for the zinc finger domain of ARFGAPs highlighted in yellow.

Bottom Line: Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability.However, silencing all three ARFGAPs causes cell death.Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 2XY, United Kingdom.

ABSTRACT
ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

Show MeSH
Related in: MedlinePlus