Limits...
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.

Show MeSH

Related in: MedlinePlus

ARFGAP2 is involved in COP-I-dependent trafficking in Vero cells. Vero cells coexpressing full-length ARFGAP2–CFP and the Golgi marker GalT-YFP were treated with CTX-K63-Cy3 (upper panel). After 3 h of internalization, CTX-K63-Cy3, the Cy3-labelled A-subunit, is prominently present in fine ER structures (including the nuclear envelope; arrow) and in the Golgi complex. ARFGAP2–CFP colocalizes well with GalT-YFP in the Golgi. Additionally, ARFGAP2–CFP is present in scattered punctate structures, most likely intermediate compartment. In contrast, in Vero cells expressing ΔN-ARFGAP2–CFP2 transport of CTX-K63-Cy3 to the ER is inhibited (middle panel). ΔN-ARFGAP2–CFP2 localizes to the Golgi complex. Vero cells overexpressing ΔN-ARFGAP2–CFP2 for extended periods often display two to three nuclei and show severe inhibition of toxin transport (lower panel). The bottom panel is the characterization of the Cy3-labelled CTX-K63 (non-toxic AB5 holotoxin) used for the experiments (for details see methods). Mostly the A-subunit of CTX-K63 is labelled, with traces of B present. Scale bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171037&req=5

fig07: ARFGAP2 is involved in COP-I-dependent trafficking in Vero cells. Vero cells coexpressing full-length ARFGAP2–CFP and the Golgi marker GalT-YFP were treated with CTX-K63-Cy3 (upper panel). After 3 h of internalization, CTX-K63-Cy3, the Cy3-labelled A-subunit, is prominently present in fine ER structures (including the nuclear envelope; arrow) and in the Golgi complex. ARFGAP2–CFP colocalizes well with GalT-YFP in the Golgi. Additionally, ARFGAP2–CFP is present in scattered punctate structures, most likely intermediate compartment. In contrast, in Vero cells expressing ΔN-ARFGAP2–CFP2 transport of CTX-K63-Cy3 to the ER is inhibited (middle panel). ΔN-ARFGAP2–CFP2 localizes to the Golgi complex. Vero cells overexpressing ΔN-ARFGAP2–CFP2 for extended periods often display two to three nuclei and show severe inhibition of toxin transport (lower panel). The bottom panel is the characterization of the Cy3-labelled CTX-K63 (non-toxic AB5 holotoxin) used for the experiments (for details see methods). Mostly the A-subunit of CTX-K63 is labelled, with traces of B present. Scale bar: 10 μm.

Mentions: Full-length ARFGAP2 was tagged at the carboxy terminus with cyan fluorescent protein (CFP) and expressed at low level in Vero cells. This CFP fusion protein localized to the Golgi complex [as identified using the Golgi enzyme marker galactosyl transferase tagged with yellow fluorescent protein (GalT-YFP)] and also localized to punctate structures scattered through the cytoplasm (Figure 7, upper panel). This localization pattern is reminiscent of the localization observed with antibodies against ARFGAP2 (compare with Figure 3).


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

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

ARFGAP2 is involved in COP-I-dependent trafficking in Vero cells. Vero cells coexpressing full-length ARFGAP2–CFP and the Golgi marker GalT-YFP were treated with CTX-K63-Cy3 (upper panel). After 3 h of internalization, CTX-K63-Cy3, the Cy3-labelled A-subunit, is prominently present in fine ER structures (including the nuclear envelope; arrow) and in the Golgi complex. ARFGAP2–CFP colocalizes well with GalT-YFP in the Golgi. Additionally, ARFGAP2–CFP is present in scattered punctate structures, most likely intermediate compartment. In contrast, in Vero cells expressing ΔN-ARFGAP2–CFP2 transport of CTX-K63-Cy3 to the ER is inhibited (middle panel). ΔN-ARFGAP2–CFP2 localizes to the Golgi complex. Vero cells overexpressing ΔN-ARFGAP2–CFP2 for extended periods often display two to three nuclei and show severe inhibition of toxin transport (lower panel). The bottom panel is the characterization of the Cy3-labelled CTX-K63 (non-toxic AB5 holotoxin) used for the experiments (for details see methods). Mostly the A-subunit of CTX-K63 is labelled, with traces of B present. Scale bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig07: ARFGAP2 is involved in COP-I-dependent trafficking in Vero cells. Vero cells coexpressing full-length ARFGAP2–CFP and the Golgi marker GalT-YFP were treated with CTX-K63-Cy3 (upper panel). After 3 h of internalization, CTX-K63-Cy3, the Cy3-labelled A-subunit, is prominently present in fine ER structures (including the nuclear envelope; arrow) and in the Golgi complex. ARFGAP2–CFP colocalizes well with GalT-YFP in the Golgi. Additionally, ARFGAP2–CFP is present in scattered punctate structures, most likely intermediate compartment. In contrast, in Vero cells expressing ΔN-ARFGAP2–CFP2 transport of CTX-K63-Cy3 to the ER is inhibited (middle panel). ΔN-ARFGAP2–CFP2 localizes to the Golgi complex. Vero cells overexpressing ΔN-ARFGAP2–CFP2 for extended periods often display two to three nuclei and show severe inhibition of toxin transport (lower panel). The bottom panel is the characterization of the Cy3-labelled CTX-K63 (non-toxic AB5 holotoxin) used for the experiments (for details see methods). Mostly the A-subunit of CTX-K63 is labelled, with traces of B present. Scale bar: 10 μm.
Mentions: Full-length ARFGAP2 was tagged at the carboxy terminus with cyan fluorescent protein (CFP) and expressed at low level in Vero cells. This CFP fusion protein localized to the Golgi complex [as identified using the Golgi enzyme marker galactosyl transferase tagged with yellow fluorescent protein (GalT-YFP)] and also localized to punctate structures scattered through the cytoplasm (Figure 7, upper panel). This localization pattern is reminiscent of the localization observed with antibodies against ARFGAP2 (compare with Figure 3).

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