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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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Silencing of all three ARFGAPs is lethal in HeLa cells. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 failed to cause lethality in HeLa cells, as well as in NRK cells (data not shown). A) The depletion of ARFGAP1, ARFGAP2 or ARFGAP3 in single knock-downs in HeLa cells was verified by immunoblotting. Antibodies against tubulin and γ-COP were used to verify equal protein loading. B and C) Cell counting was performed from DAPI-stained HeLa cells grown on coverslips. Note the strong reduction in cell numbers in the ARFGAP triple knock-down compared with a double knock-down involving ARFGAP2 and ARFGAP3 combined with a control oligo. For details of the oligos, see Materials and Methods. D) Quantification of the reduction in cell numbers from the above. More than 75% cell death was routinely observed after three sets of siRNA transfections at the 72-h time-point after the last transfection (in 10 independent experiments). Bar = 100 μm.
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fig08: Silencing of all three ARFGAPs is lethal in HeLa cells. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 failed to cause lethality in HeLa cells, as well as in NRK cells (data not shown). A) The depletion of ARFGAP1, ARFGAP2 or ARFGAP3 in single knock-downs in HeLa cells was verified by immunoblotting. Antibodies against tubulin and γ-COP were used to verify equal protein loading. B and C) Cell counting was performed from DAPI-stained HeLa cells grown on coverslips. Note the strong reduction in cell numbers in the ARFGAP triple knock-down compared with a double knock-down involving ARFGAP2 and ARFGAP3 combined with a control oligo. For details of the oligos, see Materials and Methods. D) Quantification of the reduction in cell numbers from the above. More than 75% cell death was routinely observed after three sets of siRNA transfections at the 72-h time-point after the last transfection (in 10 independent experiments). Bar = 100 μm.

Mentions: At even longer time-points after transfection (16 h), cells expressing ΔN-ARFGAP2–CFP displayed a strikingly higher frequency of having two or even three nuclei (indicated by numbers in Figure 8, lower panel). In such cells, as judged by GalT-YFP, usually only one large Golgi complex was present to which ΔN-ARFGAP2–CFP localized (Figure 7, lower panel). In such cells, transport of CTX-K63-Cy3 was again severely inhibited, with the toxin not arriving at the ER network even after 3 h. Instead, CTX-K63-Cy3 was present in the Golgi complex and large cytoplasmic structures (Figure 7, lower panel). Our data strongly suggest that ARFGAP2 is involved in the COP-I-dependent trafficking of cholera toxin from the Golgi to the ER.


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

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

Silencing of all three ARFGAPs is lethal in HeLa cells. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 failed to cause lethality in HeLa cells, as well as in NRK cells (data not shown). A) The depletion of ARFGAP1, ARFGAP2 or ARFGAP3 in single knock-downs in HeLa cells was verified by immunoblotting. Antibodies against tubulin and γ-COP were used to verify equal protein loading. B and C) Cell counting was performed from DAPI-stained HeLa cells grown on coverslips. Note the strong reduction in cell numbers in the ARFGAP triple knock-down compared with a double knock-down involving ARFGAP2 and ARFGAP3 combined with a control oligo. For details of the oligos, see Materials and Methods. D) Quantification of the reduction in cell numbers from the above. More than 75% cell death was routinely observed after three sets of siRNA transfections at the 72-h time-point after the last transfection (in 10 independent experiments). Bar = 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171037&req=5

fig08: Silencing of all three ARFGAPs is lethal in HeLa cells. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 failed to cause lethality in HeLa cells, as well as in NRK cells (data not shown). A) The depletion of ARFGAP1, ARFGAP2 or ARFGAP3 in single knock-downs in HeLa cells was verified by immunoblotting. Antibodies against tubulin and γ-COP were used to verify equal protein loading. B and C) Cell counting was performed from DAPI-stained HeLa cells grown on coverslips. Note the strong reduction in cell numbers in the ARFGAP triple knock-down compared with a double knock-down involving ARFGAP2 and ARFGAP3 combined with a control oligo. For details of the oligos, see Materials and Methods. D) Quantification of the reduction in cell numbers from the above. More than 75% cell death was routinely observed after three sets of siRNA transfections at the 72-h time-point after the last transfection (in 10 independent experiments). Bar = 100 μm.
Mentions: At even longer time-points after transfection (16 h), cells expressing ΔN-ARFGAP2–CFP displayed a strikingly higher frequency of having two or even three nuclei (indicated by numbers in Figure 8, lower panel). In such cells, as judged by GalT-YFP, usually only one large Golgi complex was present to which ΔN-ARFGAP2–CFP localized (Figure 7, lower panel). In such cells, transport of CTX-K63-Cy3 was again severely inhibited, with the toxin not arriving at the ER network even after 3 h. Instead, CTX-K63-Cy3 was present in the Golgi complex and large cytoplasmic structures (Figure 7, lower panel). Our data strongly suggest that ARFGAP2 is involved in the COP-I-dependent trafficking of cholera toxin from the Golgi to the ER.

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