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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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Sequence alignment of yeast Glo3p and human ARFGAP2 and ARFGAP3. ARFGAP2 and ARFGAP3 share 49.6% protein sequence identity. ARFGAP2 and ARFGAP3 share 17.6 and 19.9% sequence identity with Glo3p, respectively.
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fig02: Sequence alignment of yeast Glo3p and human ARFGAP2 and ARFGAP3. ARFGAP2 and ARFGAP3 share 49.6% protein sequence identity. ARFGAP2 and ARFGAP3 share 17.6 and 19.9% sequence identity with Glo3p, respectively.

Mentions: The Glo3 motif, which is always found strictly associated with an ARFGAP domain, allowed us to identify the true human orthologues of yeast Glo3p as ARFGAP2 and ARFGAP3, by sequence comparison. In Figure 2, a protein sequence alignment of yeast Glo3p, ARFGAP2 and ARFGAP3 is shown. ARFGAP2 is a protein of 521 residues and had not been previously named. However, a zinc finger protein of unknown function and designated Zfp289 had been previously identified from SCp2 mouse mammary epithelial cells (25). The protein sequence of human ARFGAP2 and mouse Zfp289 shows complete identity. ARFGAP2 and ARFGAP3 share 49.6% protein sequence identity. In an alignment over their entire length, Glo3p shares only 17.6 and 19.9% identity with ARFGAP2 and ARFGAP3, respectively. As for ARFGAP3 (516 residues), both the presence of the ARFGAP domain and its function as a GTPase-activating protein towards ARF in vitro had been noted (26,27). However, neither ARFGAP2 nor ARFGAP3 have previously been recognized as Glo3p rather than Gcs1p orthologues, and the localization of both ARFGAP2 and ARFGAP3 and their role in COP I transport has not been investigated.


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

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

Sequence alignment of yeast Glo3p and human ARFGAP2 and ARFGAP3. ARFGAP2 and ARFGAP3 share 49.6% protein sequence identity. ARFGAP2 and ARFGAP3 share 17.6 and 19.9% sequence identity with Glo3p, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig02: Sequence alignment of yeast Glo3p and human ARFGAP2 and ARFGAP3. ARFGAP2 and ARFGAP3 share 49.6% protein sequence identity. ARFGAP2 and ARFGAP3 share 17.6 and 19.9% sequence identity with Glo3p, respectively.
Mentions: The Glo3 motif, which is always found strictly associated with an ARFGAP domain, allowed us to identify the true human orthologues of yeast Glo3p as ARFGAP2 and ARFGAP3, by sequence comparison. In Figure 2, a protein sequence alignment of yeast Glo3p, ARFGAP2 and ARFGAP3 is shown. ARFGAP2 is a protein of 521 residues and had not been previously named. However, a zinc finger protein of unknown function and designated Zfp289 had been previously identified from SCp2 mouse mammary epithelial cells (25). The protein sequence of human ARFGAP2 and mouse Zfp289 shows complete identity. ARFGAP2 and ARFGAP3 share 49.6% protein sequence identity. In an alignment over their entire length, Glo3p shares only 17.6 and 19.9% identity with ARFGAP2 and ARFGAP3, respectively. As for ARFGAP3 (516 residues), both the presence of the ARFGAP domain and its function as a GTPase-activating protein towards ARF in vitro had been noted (26,27). However, neither ARFGAP2 nor ARFGAP3 have previously been recognized as Glo3p rather than Gcs1p orthologues, and the localization of both ARFGAP2 and ARFGAP3 and their role in COP I transport has not been investigated.

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