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

Direct interaction of yeast and human Glo3 with coatomer. His6- or GST-tagged proteins were used to pull down coatomer from yeast or rat liver cytosol, under conditions described by us previously (17). A) His6-tagged Glo3p lacking its amino terminus was compared with full-length Glo3p as well as full-length Gcs1p in its ability to bind coatomer from yeast cytosol. Protein bound to Ni-NTA agarose beads was resolved by SDS–PAGE, followed by Western blotting and incubation with anti-yeast coatomer antibodies detected by ECL. His-tagged Glo3p lacking its ARFGAP domain (ΔN96) still binds coatomer from yeast cytosol in vitro, whereas Gcs1p does not. GTP dissociation inhibitor is used as a negative control here. B) A GST-fusion protein harbouring ΔN96-ARFGAP2 was used for a pull-down from a centrifugation-cleared TX-100 extract of pig brain crude microsomal membranes. Glutathione S-transferase was used as the negative control. Note absence of binding in the negative control and the presence of the characteristic coatomer bands in the pull-down involving the ΔN96-ARFGAP2 GST fusion as the fishing hook. Enrichment of α- and β-COP in the bound fraction was confirmed using antipeptide antibodies (data not shown).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171037&req=5

fig06: Direct interaction of yeast and human Glo3 with coatomer. His6- or GST-tagged proteins were used to pull down coatomer from yeast or rat liver cytosol, under conditions described by us previously (17). A) His6-tagged Glo3p lacking its amino terminus was compared with full-length Glo3p as well as full-length Gcs1p in its ability to bind coatomer from yeast cytosol. Protein bound to Ni-NTA agarose beads was resolved by SDS–PAGE, followed by Western blotting and incubation with anti-yeast coatomer antibodies detected by ECL. His-tagged Glo3p lacking its ARFGAP domain (ΔN96) still binds coatomer from yeast cytosol in vitro, whereas Gcs1p does not. GTP dissociation inhibitor is used as a negative control here. B) A GST-fusion protein harbouring ΔN96-ARFGAP2 was used for a pull-down from a centrifugation-cleared TX-100 extract of pig brain crude microsomal membranes. Glutathione S-transferase was used as the negative control. Note absence of binding in the negative control and the presence of the characteristic coatomer bands in the pull-down involving the ΔN96-ARFGAP2 GST fusion as the fishing hook. Enrichment of α- and β-COP in the bound fraction was confirmed using antipeptide antibodies (data not shown).

Mentions: Yeast Glo3p interacts with γ-COP as well as β′-COP in the two-hybrid system (17). This strong, direct interaction was confirmed by pull-down experiments using recombinant His-tagged Glo3p and yeast cytosol (17). Here we show that the catalytic domain of yeast Glo3p is not required for this direct interaction with coatomer. Deletion of the N-terminal 96 amino acids including the Zn finger domain does not reduce the interaction of tagged Glo3p with coatomer in vitro (Figure 6A). In this type of experiment, Gcs1p does not bind coatomer above background levels defined by recombinant GTP dissociation inhibitor (GDI) as a negative control (Figure 6A). In order to test whether mammalian ARFGAP2 interacts with coatomer in a way comparable to yeast Glo3p, glutathione S-transferase (GST)-tagged ARFGAP2 lacking its ARFGAP domain was used for pull-down experiments from rat liver cytosol. Indeed, coatomer binding from rat liver cytosol to GST-tagged ARFGAP2 present on glutathione beads was readily detectable by silver stain (Figure 6B), and immunoblots using anti-peptide antibodies against α- and β-COP demonstrated enrichment of these coatomer subunits (data not shown). Thus, the catalytic domain of Glo3-type ARFGAP proteins from yeast and mammals is not required for in vitro interaction with coatomer.


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

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

Direct interaction of yeast and human Glo3 with coatomer. His6- or GST-tagged proteins were used to pull down coatomer from yeast or rat liver cytosol, under conditions described by us previously (17). A) His6-tagged Glo3p lacking its amino terminus was compared with full-length Glo3p as well as full-length Gcs1p in its ability to bind coatomer from yeast cytosol. Protein bound to Ni-NTA agarose beads was resolved by SDS–PAGE, followed by Western blotting and incubation with anti-yeast coatomer antibodies detected by ECL. His-tagged Glo3p lacking its ARFGAP domain (ΔN96) still binds coatomer from yeast cytosol in vitro, whereas Gcs1p does not. GTP dissociation inhibitor is used as a negative control here. B) A GST-fusion protein harbouring ΔN96-ARFGAP2 was used for a pull-down from a centrifugation-cleared TX-100 extract of pig brain crude microsomal membranes. Glutathione S-transferase was used as the negative control. Note absence of binding in the negative control and the presence of the characteristic coatomer bands in the pull-down involving the ΔN96-ARFGAP2 GST fusion as the fishing hook. Enrichment of α- and β-COP in the bound fraction was confirmed using antipeptide antibodies (data not shown).
© Copyright Policy
Related In: Results  -  Collection

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

fig06: Direct interaction of yeast and human Glo3 with coatomer. His6- or GST-tagged proteins were used to pull down coatomer from yeast or rat liver cytosol, under conditions described by us previously (17). A) His6-tagged Glo3p lacking its amino terminus was compared with full-length Glo3p as well as full-length Gcs1p in its ability to bind coatomer from yeast cytosol. Protein bound to Ni-NTA agarose beads was resolved by SDS–PAGE, followed by Western blotting and incubation with anti-yeast coatomer antibodies detected by ECL. His-tagged Glo3p lacking its ARFGAP domain (ΔN96) still binds coatomer from yeast cytosol in vitro, whereas Gcs1p does not. GTP dissociation inhibitor is used as a negative control here. B) A GST-fusion protein harbouring ΔN96-ARFGAP2 was used for a pull-down from a centrifugation-cleared TX-100 extract of pig brain crude microsomal membranes. Glutathione S-transferase was used as the negative control. Note absence of binding in the negative control and the presence of the characteristic coatomer bands in the pull-down involving the ΔN96-ARFGAP2 GST fusion as the fishing hook. Enrichment of α- and β-COP in the bound fraction was confirmed using antipeptide antibodies (data not shown).
Mentions: Yeast Glo3p interacts with γ-COP as well as β′-COP in the two-hybrid system (17). This strong, direct interaction was confirmed by pull-down experiments using recombinant His-tagged Glo3p and yeast cytosol (17). Here we show that the catalytic domain of yeast Glo3p is not required for this direct interaction with coatomer. Deletion of the N-terminal 96 amino acids including the Zn finger domain does not reduce the interaction of tagged Glo3p with coatomer in vitro (Figure 6A). In this type of experiment, Gcs1p does not bind coatomer above background levels defined by recombinant GTP dissociation inhibitor (GDI) as a negative control (Figure 6A). In order to test whether mammalian ARFGAP2 interacts with coatomer in a way comparable to yeast Glo3p, glutathione S-transferase (GST)-tagged ARFGAP2 lacking its ARFGAP domain was used for pull-down experiments from rat liver cytosol. Indeed, coatomer binding from rat liver cytosol to GST-tagged ARFGAP2 present on glutathione beads was readily detectable by silver stain (Figure 6B), and immunoblots using anti-peptide antibodies against α- and β-COP demonstrated enrichment of these coatomer subunits (data not shown). Thus, the catalytic domain of Glo3-type ARFGAP proteins from yeast and mammals is not required for in vitro interaction with coatomer.

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