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GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5.

Claude A, Zhao BP, Kuziemsky CE, Dahan S, Berger SJ, Yan JP, Armold AD, Sullivan EM, Melançon P - J. Cell Biol. (1999)

Bottom Line: GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI.Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures.The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.

ABSTRACT
Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

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(His)6-GBF1 is a BFA-resistant ARF-GEF. (A) Fractions enriched in (His)6-GBF1 display a GEF specific for ARFs. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (vector alone) and (His)6GBF1 extracts were assayed for loading of small GTPases with GTPγS in 1 μM of free Mg2+ as described in Materials and Methods. Purified ARF1/3, Sar1p, or rab1b (1 μM each) were used as substrate as indicated. Each bar represents the average of three determinations ± SD. (B) GEF activity of GBF1 towards ARF1/3 is BFA resistant. Time course of ARF1/3 nucleotide exchange using the 50 mM imidazole eluate fraction from the (His)6GBF1 extracts. Assays were performed at 1 μM of free Mg2+ either in the presence (closed circle) or absence (open circle) of BFA (360 μM). Similar results were obtained with several independent preparations. (C) The purified Sec7 domain of Sec7 is a BFA-sensitive ARF-GEF. Assays were performed as described in Materials and Methods with the indicated amounts of BFA. Each bar represents the average of three determinations ± SD. (D) GBF1 is a BFA-resistant GEF specific for ARF5 at 1 mM Mg2+. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (empty vector) and (His)6GBF1 extracts were assayed for loading of ARF1/3 or ARF5 in assays containing 1 mM Mg2+. BFA was used at a concentration of 600 μM. One set of reactions was performed with the GBF1 fraction inactivated (asterisk) by incubation at 95°C for 5 min. Each bar represents the average of three determinations ± SD.
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Figure 6: (His)6-GBF1 is a BFA-resistant ARF-GEF. (A) Fractions enriched in (His)6-GBF1 display a GEF specific for ARFs. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (vector alone) and (His)6GBF1 extracts were assayed for loading of small GTPases with GTPγS in 1 μM of free Mg2+ as described in Materials and Methods. Purified ARF1/3, Sar1p, or rab1b (1 μM each) were used as substrate as indicated. Each bar represents the average of three determinations ± SD. (B) GEF activity of GBF1 towards ARF1/3 is BFA resistant. Time course of ARF1/3 nucleotide exchange using the 50 mM imidazole eluate fraction from the (His)6GBF1 extracts. Assays were performed at 1 μM of free Mg2+ either in the presence (closed circle) or absence (open circle) of BFA (360 μM). Similar results were obtained with several independent preparations. (C) The purified Sec7 domain of Sec7 is a BFA-sensitive ARF-GEF. Assays were performed as described in Materials and Methods with the indicated amounts of BFA. Each bar represents the average of three determinations ± SD. (D) GBF1 is a BFA-resistant GEF specific for ARF5 at 1 mM Mg2+. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (empty vector) and (His)6GBF1 extracts were assayed for loading of ARF1/3 or ARF5 in assays containing 1 mM Mg2+. BFA was used at a concentration of 600 μM. One set of reactions was performed with the GBF1 fraction inactivated (asterisk) by incubation at 95°C for 5 min. Each bar represents the average of three determinations ± SD.

Mentions: To confirm that GBF1 had ARF-GEF activity and determine whether this activity was sensitive to BFA, we modified GBF1 with a hexahistidine tag to facilitate its purification. Control transfection experiments established that tagging did not reduce the ability of GBF1 to cause BFA resistance (not shown). A significant fraction of GBF1 from detergent extracts of (His)6-GBF1 transformants bound Ni-NTA columns and eluted at a 50 mM imidazole concentration (not shown). In contrast, endogenous GBF1 in extracts from control cells remained in the flowthrough fraction. At 1 μM of free Mg2+, eluate fractions containing tagged-GBF1 stimulated binding of GTP on native ARFs from the bovine brain (1:9 mixture of ARF1 and ARF3), whereas those from control cells showed no activity (Fig. 6 A). This GEF activity appears specific for small GTPases of the ARF family since no such stimulation was observed with purified Sar1p or rab1b (Fig. 6 A). The GEF activity observed on ARF1/3 is significant and clearly above the spontaneous loading observed with ARF alone. This background was much lower than previously reported by Paris et al. 1997 for recombinant myristoylated ARF1 that indicates that ARF3 spontaneously binds GTP at a much lower rate than ARF1 at low Mg2+ concentrations.


GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5.

Claude A, Zhao BP, Kuziemsky CE, Dahan S, Berger SJ, Yan JP, Armold AD, Sullivan EM, Melançon P - J. Cell Biol. (1999)

(His)6-GBF1 is a BFA-resistant ARF-GEF. (A) Fractions enriched in (His)6-GBF1 display a GEF specific for ARFs. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (vector alone) and (His)6GBF1 extracts were assayed for loading of small GTPases with GTPγS in 1 μM of free Mg2+ as described in Materials and Methods. Purified ARF1/3, Sar1p, or rab1b (1 μM each) were used as substrate as indicated. Each bar represents the average of three determinations ± SD. (B) GEF activity of GBF1 towards ARF1/3 is BFA resistant. Time course of ARF1/3 nucleotide exchange using the 50 mM imidazole eluate fraction from the (His)6GBF1 extracts. Assays were performed at 1 μM of free Mg2+ either in the presence (closed circle) or absence (open circle) of BFA (360 μM). Similar results were obtained with several independent preparations. (C) The purified Sec7 domain of Sec7 is a BFA-sensitive ARF-GEF. Assays were performed as described in Materials and Methods with the indicated amounts of BFA. Each bar represents the average of three determinations ± SD. (D) GBF1 is a BFA-resistant GEF specific for ARF5 at 1 mM Mg2+. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (empty vector) and (His)6GBF1 extracts were assayed for loading of ARF1/3 or ARF5 in assays containing 1 mM Mg2+. BFA was used at a concentration of 600 μM. One set of reactions was performed with the GBF1 fraction inactivated (asterisk) by incubation at 95°C for 5 min. Each bar represents the average of three determinations ± SD.
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Figure 6: (His)6-GBF1 is a BFA-resistant ARF-GEF. (A) Fractions enriched in (His)6-GBF1 display a GEF specific for ARFs. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (vector alone) and (His)6GBF1 extracts were assayed for loading of small GTPases with GTPγS in 1 μM of free Mg2+ as described in Materials and Methods. Purified ARF1/3, Sar1p, or rab1b (1 μM each) were used as substrate as indicated. Each bar represents the average of three determinations ± SD. (B) GEF activity of GBF1 towards ARF1/3 is BFA resistant. Time course of ARF1/3 nucleotide exchange using the 50 mM imidazole eluate fraction from the (His)6GBF1 extracts. Assays were performed at 1 μM of free Mg2+ either in the presence (closed circle) or absence (open circle) of BFA (360 μM). Similar results were obtained with several independent preparations. (C) The purified Sec7 domain of Sec7 is a BFA-sensitive ARF-GEF. Assays were performed as described in Materials and Methods with the indicated amounts of BFA. Each bar represents the average of three determinations ± SD. (D) GBF1 is a BFA-resistant GEF specific for ARF5 at 1 mM Mg2+. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (empty vector) and (His)6GBF1 extracts were assayed for loading of ARF1/3 or ARF5 in assays containing 1 mM Mg2+. BFA was used at a concentration of 600 μM. One set of reactions was performed with the GBF1 fraction inactivated (asterisk) by incubation at 95°C for 5 min. Each bar represents the average of three determinations ± SD.
Mentions: To confirm that GBF1 had ARF-GEF activity and determine whether this activity was sensitive to BFA, we modified GBF1 with a hexahistidine tag to facilitate its purification. Control transfection experiments established that tagging did not reduce the ability of GBF1 to cause BFA resistance (not shown). A significant fraction of GBF1 from detergent extracts of (His)6-GBF1 transformants bound Ni-NTA columns and eluted at a 50 mM imidazole concentration (not shown). In contrast, endogenous GBF1 in extracts from control cells remained in the flowthrough fraction. At 1 μM of free Mg2+, eluate fractions containing tagged-GBF1 stimulated binding of GTP on native ARFs from the bovine brain (1:9 mixture of ARF1 and ARF3), whereas those from control cells showed no activity (Fig. 6 A). This GEF activity appears specific for small GTPases of the ARF family since no such stimulation was observed with purified Sar1p or rab1b (Fig. 6 A). The GEF activity observed on ARF1/3 is significant and clearly above the spontaneous loading observed with ARF alone. This background was much lower than previously reported by Paris et al. 1997 for recombinant myristoylated ARF1 that indicates that ARF3 spontaneously binds GTP at a much lower rate than ARF1 at low Mg2+ concentrations.

Bottom Line: GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI.Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures.The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.

ABSTRACT
Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

Show MeSH
Related in: MedlinePlus