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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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The Golgi morphology of 293 overexpressing GBF1 is resistant to BFA. 293 cells were transformed as described with pCEP4 vector (control) or pCEP4-GBF1 (GBF1) and grown on coverslips. The cells were incubated in the absence (A and B) or in the presence of 4 μM BFA (C and D) for 20 min before fixation and staining with antigiantin serum as described in Materials and Methods. Bar, 10 μm.
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Figure 3: The Golgi morphology of 293 overexpressing GBF1 is resistant to BFA. 293 cells were transformed as described with pCEP4 vector (control) or pCEP4-GBF1 (GBF1) and grown on coverslips. The cells were incubated in the absence (A and B) or in the presence of 4 μM BFA (C and D) for 20 min before fixation and staining with antigiantin serum as described in Materials and Methods. Bar, 10 μm.

Mentions: To determine if the growth advantage resulting from GBF1 expression correlated with stabilization of the Golgi complex, we tested the effect of BFA on the distribution of Golgi markers in control and GBF1-expressing cells. Such stable GBF1 transformants grew at BFA concentrations 10–15-fold higher than control transformants containing empty vector and maintained this level of resistance for at least 4 mo of continuous culture. Giantin, a well characterized Golgi complex marker (Linstedt and Hauri 1993) was used to evaluate the morphology of GBF1 transformed cells. As shown in Fig. 3, expression of GBF1 allowed cells to maintain the characteristic perinuclear localization of their Golgi complex in the presence of 4 μM BFA, a concentration that led to complete dispersal in control cells.


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)

The Golgi morphology of 293 overexpressing GBF1 is resistant to BFA. 293 cells were transformed as described with pCEP4 vector (control) or pCEP4-GBF1 (GBF1) and grown on coverslips. The cells were incubated in the absence (A and B) or in the presence of 4 μM BFA (C and D) for 20 min before fixation and staining with antigiantin serum as described in Materials and Methods. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199737&req=5

Figure 3: The Golgi morphology of 293 overexpressing GBF1 is resistant to BFA. 293 cells were transformed as described with pCEP4 vector (control) or pCEP4-GBF1 (GBF1) and grown on coverslips. The cells were incubated in the absence (A and B) or in the presence of 4 μM BFA (C and D) for 20 min before fixation and staining with antigiantin serum as described in Materials and Methods. Bar, 10 μm.
Mentions: To determine if the growth advantage resulting from GBF1 expression correlated with stabilization of the Golgi complex, we tested the effect of BFA on the distribution of Golgi markers in control and GBF1-expressing cells. Such stable GBF1 transformants grew at BFA concentrations 10–15-fold higher than control transformants containing empty vector and maintained this level of resistance for at least 4 mo of continuous culture. Giantin, a well characterized Golgi complex marker (Linstedt and Hauri 1993) was used to evaluate the morphology of GBF1 transformed cells. As shown in Fig. 3, expression of GBF1 allowed cells to maintain the characteristic perinuclear localization of their Golgi complex in the presence of 4 μM BFA, a concentration that led to complete dispersal in control cells.

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