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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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ARFGAP2 and ARFGAP3 are associated with COP I vesicles generated in vitro. Vesicle budding reactions were performed according to the protocol developed by (31), using the non-hydrolysable GTP analogue, GTP-γ-S. Vesicles produced in the reaction were separated from the Golgi donor membranes on sucrose density gradients and peaked at 40–43% sucrose. Fractions were analysed by silver stain (upper panel) and immunoblotting with antibodies as indicated (lower panel). The positions of coatomer bands are indicated on the left and also by asterisks. Note the abundant presence of ARFGAP2 and ARFGAP3 in the vesicle fractions defined by the presence of coatomer subunits (fractions 8 + 9) but the absence of ARFGAP1.
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fig05: ARFGAP2 and ARFGAP3 are associated with COP I vesicles generated in vitro. Vesicle budding reactions were performed according to the protocol developed by (31), using the non-hydrolysable GTP analogue, GTP-γ-S. Vesicles produced in the reaction were separated from the Golgi donor membranes on sucrose density gradients and peaked at 40–43% sucrose. Fractions were analysed by silver stain (upper panel) and immunoblotting with antibodies as indicated (lower panel). The positions of coatomer bands are indicated on the left and also by asterisks. Note the abundant presence of ARFGAP2 and ARFGAP3 in the vesicle fractions defined by the presence of coatomer subunits (fractions 8 + 9) but the absence of ARFGAP1.

Mentions: We next wished to test whether ARFGAP2 and ARFGAP3 are associated with COP I vesicles produced in vitro. For this we employed the ‘classic’ budding assay developed by the Rothman/Wieland labs (31,32), using purified rat liver Golgi and pig brain cytosol. The budding reaction was performed in the presence of the nonhydrolysable GTP analogue, GTP-γ-S, which locks ARF on the membrane of vesicles and thus prevents uncoating. Vesicles and Golgi donor membranes were separated on a linear sucrose gradient by overnight centrifugation (see Materials and Methods, and 31), and proteins in the fractions obtained were analysed by immunoblotting and silver staining of bands after SDS–PAGE (Figure 5). The characteristic set of coatomer bands (α-, β’-, β-, γ- and δ-COP) were found enriched in the expected positions for COP-I-coated vesicles in the gradient (fractions 8 + 9; corresponding to 40–43% sucrose) where also the blot signals for γ-COP (Figure 5A,B) and β-COP (data not shown) showed a corresponding major peak. ARFGAP1 was predominantly detected in the donor Golgi fractions, but only small amounts were found in the fractions containing COP I vesicles (Figure 5B). This is consistent with the previously reported findings from the Hsu lab that ARFGAP1 is depleted from COP I vesicles formed in the presence of GTP-γ-S (23). On the other hand, ARFGAP2 and ARFGAP3 showed a strong peak in the COP I vesicle fractions (Figure 5B), indicating that these novel Glo3-type ARFGAPs can be actively recruited into budding COP I vesicles even in the presence of GTP-γ-S. Clathrin heavy chain and the γ-subunit of the AP-1 adaptor complex used as controls were absent from the COP I vesicle fractions, as expected. We find that the dilysine motif-bearing protein ERGIC-53 was also not included into the COP I vesicles. ADP-ribosylation factor-1, which is involved in many different transport steps within the Golgi complex, was found both in the donor Golgi fractions and in the COP I vesicle fractions, as expected. Our data show that both novel ARFGAPs, ARFGAP2 and ARFGAP3 are associated with the COP-I-coated vesicles produced in vitro in the presence of GTPγS, whereas ARFGAP1 is not or much less so.


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

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

ARFGAP2 and ARFGAP3 are associated with COP I vesicles generated in vitro. Vesicle budding reactions were performed according to the protocol developed by (31), using the non-hydrolysable GTP analogue, GTP-γ-S. Vesicles produced in the reaction were separated from the Golgi donor membranes on sucrose density gradients and peaked at 40–43% sucrose. Fractions were analysed by silver stain (upper panel) and immunoblotting with antibodies as indicated (lower panel). The positions of coatomer bands are indicated on the left and also by asterisks. Note the abundant presence of ARFGAP2 and ARFGAP3 in the vesicle fractions defined by the presence of coatomer subunits (fractions 8 + 9) but the absence of ARFGAP1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171037&req=5

fig05: ARFGAP2 and ARFGAP3 are associated with COP I vesicles generated in vitro. Vesicle budding reactions were performed according to the protocol developed by (31), using the non-hydrolysable GTP analogue, GTP-γ-S. Vesicles produced in the reaction were separated from the Golgi donor membranes on sucrose density gradients and peaked at 40–43% sucrose. Fractions were analysed by silver stain (upper panel) and immunoblotting with antibodies as indicated (lower panel). The positions of coatomer bands are indicated on the left and also by asterisks. Note the abundant presence of ARFGAP2 and ARFGAP3 in the vesicle fractions defined by the presence of coatomer subunits (fractions 8 + 9) but the absence of ARFGAP1.
Mentions: We next wished to test whether ARFGAP2 and ARFGAP3 are associated with COP I vesicles produced in vitro. For this we employed the ‘classic’ budding assay developed by the Rothman/Wieland labs (31,32), using purified rat liver Golgi and pig brain cytosol. The budding reaction was performed in the presence of the nonhydrolysable GTP analogue, GTP-γ-S, which locks ARF on the membrane of vesicles and thus prevents uncoating. Vesicles and Golgi donor membranes were separated on a linear sucrose gradient by overnight centrifugation (see Materials and Methods, and 31), and proteins in the fractions obtained were analysed by immunoblotting and silver staining of bands after SDS–PAGE (Figure 5). The characteristic set of coatomer bands (α-, β’-, β-, γ- and δ-COP) were found enriched in the expected positions for COP-I-coated vesicles in the gradient (fractions 8 + 9; corresponding to 40–43% sucrose) where also the blot signals for γ-COP (Figure 5A,B) and β-COP (data not shown) showed a corresponding major peak. ARFGAP1 was predominantly detected in the donor Golgi fractions, but only small amounts were found in the fractions containing COP I vesicles (Figure 5B). This is consistent with the previously reported findings from the Hsu lab that ARFGAP1 is depleted from COP I vesicles formed in the presence of GTP-γ-S (23). On the other hand, ARFGAP2 and ARFGAP3 showed a strong peak in the COP I vesicle fractions (Figure 5B), indicating that these novel Glo3-type ARFGAPs can be actively recruited into budding COP I vesicles even in the presence of GTP-γ-S. Clathrin heavy chain and the γ-subunit of the AP-1 adaptor complex used as controls were absent from the COP I vesicle fractions, as expected. We find that the dilysine motif-bearing protein ERGIC-53 was also not included into the COP I vesicles. ADP-ribosylation factor-1, which is involved in many different transport steps within the Golgi complex, was found both in the donor Golgi fractions and in the COP I vesicle fractions, as expected. Our data show that both novel ARFGAPs, ARFGAP2 and ARFGAP3 are associated with the COP-I-coated vesicles produced in vitro in the presence of GTPγS, whereas ARFGAP1 is not or much less so.

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