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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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Isolation of a cDNA clone that allows cell growth in the presence of BFA. 293 cells were transformed as described in Materials and Methods with pCEP4 (empty vector), a twice-enriched BFY1 cDNA library, a pool of 33 plasmids derived from the twice-enriched cDNA library, clone 10 and clone 32 (both included in the pool of 33 plasmids). Transformants were selected and grown in the presence of 0.4 μM (0.1 μg/ml) BFA for 6 d. Cell growth was scored by light microscopy and photographs were taken at the indicated intervals.
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Figure 1: Isolation of a cDNA clone that allows cell growth in the presence of BFA. 293 cells were transformed as described in Materials and Methods with pCEP4 (empty vector), a twice-enriched BFY1 cDNA library, a pool of 33 plasmids derived from the twice-enriched cDNA library, clone 10 and clone 32 (both included in the pool of 33 plasmids). Transformants were selected and grown in the presence of 0.4 μM (0.1 μg/ml) BFA for 6 d. Cell growth was scored by light microscopy and photographs were taken at the indicated intervals.

Mentions: Transformation of 293 cells under standardized conditions with the BFY-1 cDNA library yielded stable transformants able to grow in the presence of 0.2 μM BFA at a frequency of ∼10 ± 3 per 106 hygromycin-resistant transformants, significantly higher than observed in parallel transfections with empty vector. Transformation of pooled plasmids recovered from BFA-resistant colonies grown to confluence (enriched libraries) yielded BFA-resistant transformants at a frequency of 1.4 × 103 ± 0.2 per 106 hygromycin-resistant transformants, indicating a greater than 100-fold enrichment in plasmids able to promote BFA resistance (see Materials and Methods for details). These results confirmed the presence of cDNAs encoding GBFs in the BFY-1 library and demonstrated the effectiveness of our selection method. Transformation with a twice-selected library yielded a very large number of BFA-resistant transformants (Fig. 1). Screening of 100 plasmids recovered from this library, first in pools then singly, yielded clone 32. Its insert of 6.8-kb insert was designated GBF1 (Fig. 1; see Materials and Methods for details).


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)

Isolation of a cDNA clone that allows cell growth in the presence of BFA. 293 cells were transformed as described in Materials and Methods with pCEP4 (empty vector), a twice-enriched BFY1 cDNA library, a pool of 33 plasmids derived from the twice-enriched cDNA library, clone 10 and clone 32 (both included in the pool of 33 plasmids). Transformants were selected and grown in the presence of 0.4 μM (0.1 μg/ml) BFA for 6 d. Cell growth was scored by light microscopy and photographs were taken at the indicated intervals.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Isolation of a cDNA clone that allows cell growth in the presence of BFA. 293 cells were transformed as described in Materials and Methods with pCEP4 (empty vector), a twice-enriched BFY1 cDNA library, a pool of 33 plasmids derived from the twice-enriched cDNA library, clone 10 and clone 32 (both included in the pool of 33 plasmids). Transformants were selected and grown in the presence of 0.4 μM (0.1 μg/ml) BFA for 6 d. Cell growth was scored by light microscopy and photographs were taken at the indicated intervals.
Mentions: Transformation of 293 cells under standardized conditions with the BFY-1 cDNA library yielded stable transformants able to grow in the presence of 0.2 μM BFA at a frequency of ∼10 ± 3 per 106 hygromycin-resistant transformants, significantly higher than observed in parallel transfections with empty vector. Transformation of pooled plasmids recovered from BFA-resistant colonies grown to confluence (enriched libraries) yielded BFA-resistant transformants at a frequency of 1.4 × 103 ± 0.2 per 106 hygromycin-resistant transformants, indicating a greater than 100-fold enrichment in plasmids able to promote BFA resistance (see Materials and Methods for details). These results confirmed the presence of cDNAs encoding GBFs in the BFY-1 library and demonstrated the effectiveness of our selection method. Transformation with a twice-selected library yielded a very large number of BFA-resistant transformants (Fig. 1). Screening of 100 plasmids recovered from this library, first in pools then singly, yielded clone 32. Its insert of 6.8-kb insert was designated GBF1 (Fig. 1; see Materials and Methods for details).

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