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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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Related in: MedlinePlus

ARFGAP2 colocalizes with coatomer in NRK cells. Methanol–acetone-fixed NRK cells were stained with affinity-purified ARFGAP2 antibodies and monoclonal antibodies against β-COP, p115, GM130 or γ-adaptin. Cy3-coupled anti-rabbit and Alexa488-coupled anti-mouse secondary antibodies were used. Images were acquired using a Zeiss Axioplan Fluorescent Microscope equipped with a charge-coupled device camera. The individual channels are shown in monochrome; the composite overlay is shown to the right (green channel, ARFGAP2; red channel, β-COP, p115, GM130 and γAP, respectively). Bar = 10 μm.
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fig03: ARFGAP2 colocalizes with coatomer in NRK cells. Methanol–acetone-fixed NRK cells were stained with affinity-purified ARFGAP2 antibodies and monoclonal antibodies against β-COP, p115, GM130 or γ-adaptin. Cy3-coupled anti-rabbit and Alexa488-coupled anti-mouse secondary antibodies were used. Images were acquired using a Zeiss Axioplan Fluorescent Microscope equipped with a charge-coupled device camera. The individual channels are shown in monochrome; the composite overlay is shown to the right (green channel, ARFGAP2; red channel, β-COP, p115, GM130 and γAP, respectively). Bar = 10 μm.

Mentions: In order to confirm that these two human Glo3p orthologues are involved in COP I transport, we raised monospecific polyclonal antibodies against them (see Materials and Methods) and determined their intracellular localization. By IF studies on methanol–acetone-fixed NRK cells, we find the majority of ARFGAP2 as well as ARFGAP3 associated with a juxtanuclear densely packed structure typical for the mammalian Golgi complex (Figures 3 and 4). The remaining protein is associated with small punctate structures scattered throughout the cytoplasm. Double-labelling experiments reveal that these structures labelled with antibodies against ARFGAP2 as well as ARFGAP3 are largely identical to those labelled with antibodies against subunits of the coatomer complex, identifying them as ER-Golgi intermediate compartment (ERGIC)/vesicular-tubular compartment (VTC) structures.


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

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

ARFGAP2 colocalizes with coatomer in NRK cells. Methanol–acetone-fixed NRK cells were stained with affinity-purified ARFGAP2 antibodies and monoclonal antibodies against β-COP, p115, GM130 or γ-adaptin. Cy3-coupled anti-rabbit and Alexa488-coupled anti-mouse secondary antibodies were used. Images were acquired using a Zeiss Axioplan Fluorescent Microscope equipped with a charge-coupled device camera. The individual channels are shown in monochrome; the composite overlay is shown to the right (green channel, ARFGAP2; red channel, β-COP, p115, GM130 and γAP, respectively). Bar = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig03: ARFGAP2 colocalizes with coatomer in NRK cells. Methanol–acetone-fixed NRK cells were stained with affinity-purified ARFGAP2 antibodies and monoclonal antibodies against β-COP, p115, GM130 or γ-adaptin. Cy3-coupled anti-rabbit and Alexa488-coupled anti-mouse secondary antibodies were used. Images were acquired using a Zeiss Axioplan Fluorescent Microscope equipped with a charge-coupled device camera. The individual channels are shown in monochrome; the composite overlay is shown to the right (green channel, ARFGAP2; red channel, β-COP, p115, GM130 and γAP, respectively). Bar = 10 μm.
Mentions: In order to confirm that these two human Glo3p orthologues are involved in COP I transport, we raised monospecific polyclonal antibodies against them (see Materials and Methods) and determined their intracellular localization. By IF studies on methanol–acetone-fixed NRK cells, we find the majority of ARFGAP2 as well as ARFGAP3 associated with a juxtanuclear densely packed structure typical for the mammalian Golgi complex (Figures 3 and 4). The remaining protein is associated with small punctate structures scattered throughout the cytoplasm. Double-labelling experiments reveal that these structures labelled with antibodies against ARFGAP2 as well as ARFGAP3 are largely identical to those labelled with antibodies against subunits of the coatomer complex, identifying them as ER-Golgi intermediate compartment (ERGIC)/vesicular-tubular compartment (VTC) structures.

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