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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 colocalizes with β-COP to the Golgi complex. (A) Wild-type CHO (left), mutant BFY-1 (center), and NRK (right) cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and stained with a Texas red–conjugated secondary antibody. Images obtained by standard epifluorescence microscopy are presented. (B) NRK cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and mAb m3A5 (anti–β-COP) and stained with FITC- (β-COP) and Texas red– (GBF1) conjugated secondary antibodies. The images presented were obtained by confocal microscopy. (Left) GBF1 signal; (right) β-COP signal; and (center) merge of both images. Bars, 10 μm.
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Figure 8: GBF1 colocalizes with β-COP to the Golgi complex. (A) Wild-type CHO (left), mutant BFY-1 (center), and NRK (right) cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and stained with a Texas red–conjugated secondary antibody. Images obtained by standard epifluorescence microscopy are presented. (B) NRK cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and mAb m3A5 (anti–β-COP) and stained with FITC- (β-COP) and Texas red– (GBF1) conjugated secondary antibodies. The images presented were obtained by confocal microscopy. (Left) GBF1 signal; (right) β-COP signal; and (center) merge of both images. Bars, 10 μm.

Mentions: The observation that GBF1 is a BFA-resistant ARF-GEF whose expression allows BFA-resistant recruitment of the COPI coat onto Golgi membranes strongly implicates it in protein traffic at the Golgi complex. To determine if GBF1 associates with the Golgi complex in vivo, we examined its intracellular distribution in CHO and NRK cells using indirect immunofluorescence with the H154 antiserum (Fig. 8).


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 colocalizes with β-COP to the Golgi complex. (A) Wild-type CHO (left), mutant BFY-1 (center), and NRK (right) cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and stained with a Texas red–conjugated secondary antibody. Images obtained by standard epifluorescence microscopy are presented. (B) NRK cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and mAb m3A5 (anti–β-COP) and stained with FITC- (β-COP) and Texas red– (GBF1) conjugated secondary antibodies. The images presented were obtained by confocal microscopy. (Left) GBF1 signal; (right) β-COP signal; and (center) merge of both images. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 8: GBF1 colocalizes with β-COP to the Golgi complex. (A) Wild-type CHO (left), mutant BFY-1 (center), and NRK (right) cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and stained with a Texas red–conjugated secondary antibody. Images obtained by standard epifluorescence microscopy are presented. (B) NRK cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and mAb m3A5 (anti–β-COP) and stained with FITC- (β-COP) and Texas red– (GBF1) conjugated secondary antibodies. The images presented were obtained by confocal microscopy. (Left) GBF1 signal; (right) β-COP signal; and (center) merge of both images. Bars, 10 μm.
Mentions: The observation that GBF1 is a BFA-resistant ARF-GEF whose expression allows BFA-resistant recruitment of the COPI coat onto Golgi membranes strongly implicates it in protein traffic at the Golgi complex. To determine if GBF1 associates with the Golgi complex in vivo, we examined its intracellular distribution in CHO and NRK cells using indirect immunofluorescence with the H154 antiserum (Fig. 8).

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