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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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GBF1 localizes primarily to a tubular network proximal to Golgi stacks. Rat liver frozen cryosections were processed, labeled, and photographed as described in Materials and Methods. (A) anti-GBF1 serum H134 (diluted 1:2). (B) anti-GBF1 serum H133 (diluted 1:5). (C and D) mouse anti–β-COP (diluted 1:20). (Arrowheads) Gold particles labeling ER cisternae. (Arrows) Gold particles labeling tubular networks proximal to the Golgi complex. (Curved arrows) Gold particles labeling Golgi stacks. (Brackets) High concentration of gold particles labeling tubular networks proximal to the Golgi complex. M, mitochondria; P, peroxisome; and G, Golgi stack. Bars, 400 nm.
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Figure 9: GBF1 localizes primarily to a tubular network proximal to Golgi stacks. Rat liver frozen cryosections were processed, labeled, and photographed as described in Materials and Methods. (A) anti-GBF1 serum H134 (diluted 1:2). (B) anti-GBF1 serum H133 (diluted 1:5). (C and D) mouse anti–β-COP (diluted 1:20). (Arrowheads) Gold particles labeling ER cisternae. (Arrows) Gold particles labeling tubular networks proximal to the Golgi complex. (Curved arrows) Gold particles labeling Golgi stacks. (Brackets) High concentration of gold particles labeling tubular networks proximal to the Golgi complex. M, mitochondria; P, peroxisome; and G, Golgi stack. Bars, 400 nm.

Mentions: More detailed subcellular localization of GBF1 was obtained by immunoelectron microscopy. Immunolabeling of liver ultrathin cryosections was performed with several sera raised against the COOH-terminal peptide of GBF1. All sera showed similar staining of tubular elements adjacent to Golgi stacks (Fig. 9A and Fig. B, brackets and arrows). These elements correspond to the regions of greatest antigenicity of COPI in rat liver sections (Fig. 9C and Fig. D; Dahan, S., and J.J.M. Bergeron, unpublished observations). Significant labeling was also observed over Golgi stacks, particularly at the electron lucent distensions (curved arrows), and on ER cisternae (arrowheads). Little staining of mitochondria and peroxisomes was observed under these conditions. Quantitative analysis of the GBF1 labeling experiments confirmed that even though a significant amount of GBF1 staining localized to peripheral tubules, the greatest concentration occurred in the Golgi region (Table ).


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)

GBF1 localizes primarily to a tubular network proximal to Golgi stacks. Rat liver frozen cryosections were processed, labeled, and photographed as described in Materials and Methods. (A) anti-GBF1 serum H134 (diluted 1:2). (B) anti-GBF1 serum H133 (diluted 1:5). (C and D) mouse anti–β-COP (diluted 1:20). (Arrowheads) Gold particles labeling ER cisternae. (Arrows) Gold particles labeling tubular networks proximal to the Golgi complex. (Curved arrows) Gold particles labeling Golgi stacks. (Brackets) High concentration of gold particles labeling tubular networks proximal to the Golgi complex. M, mitochondria; P, peroxisome; and G, Golgi stack. Bars, 400 nm.
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Related In: Results  -  Collection

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Figure 9: GBF1 localizes primarily to a tubular network proximal to Golgi stacks. Rat liver frozen cryosections were processed, labeled, and photographed as described in Materials and Methods. (A) anti-GBF1 serum H134 (diluted 1:2). (B) anti-GBF1 serum H133 (diluted 1:5). (C and D) mouse anti–β-COP (diluted 1:20). (Arrowheads) Gold particles labeling ER cisternae. (Arrows) Gold particles labeling tubular networks proximal to the Golgi complex. (Curved arrows) Gold particles labeling Golgi stacks. (Brackets) High concentration of gold particles labeling tubular networks proximal to the Golgi complex. M, mitochondria; P, peroxisome; and G, Golgi stack. Bars, 400 nm.
Mentions: More detailed subcellular localization of GBF1 was obtained by immunoelectron microscopy. Immunolabeling of liver ultrathin cryosections was performed with several sera raised against the COOH-terminal peptide of GBF1. All sera showed similar staining of tubular elements adjacent to Golgi stacks (Fig. 9A and Fig. B, brackets and arrows). These elements correspond to the regions of greatest antigenicity of COPI in rat liver sections (Fig. 9C and Fig. D; Dahan, S., and J.J.M. Bergeron, unpublished observations). Significant labeling was also observed over Golgi stacks, particularly at the electron lucent distensions (curved arrows), and on ER cisternae (arrowheads). Little staining of mitochondria and peroxisomes was observed under these conditions. Quantitative analysis of the GBF1 labeling experiments confirmed that even though a significant amount of GBF1 staining localized to peripheral tubules, the greatest concentration occurred in the Golgi region (Table ).

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