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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 is a naturally BFA-resistant GEF that is not overexpressed in BFY-1 cells. (A) Schematic representation of the GBF1 allele identified in wild-type CHO cells. The position of the Sec7d is indicated by the dark rectangle and the deletions by thin vertical lines. The size of the deletions and the position of the last nucleotide before each deletion are indicated above and below, respectively. These in frame deletions lead to the loss of S (622) and VSQD (1494-1497), respectively. (B) GBF1 is expressed at similar levels in wild-type CHO and mutant BFY-1 cell lines. Identical amounts of Triton X-100 extracts (30 μg) from the indicated cell lines were analyzed by PAGE/immunoblotting as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 and anti-G6PDH antibodies. The ratios of GBF1 to G6PDH were identical in both extracts.
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Figure 7: GBF1 is a naturally BFA-resistant GEF that is not overexpressed in BFY-1 cells. (A) Schematic representation of the GBF1 allele identified in wild-type CHO cells. The position of the Sec7d is indicated by the dark rectangle and the deletions by thin vertical lines. The size of the deletions and the position of the last nucleotide before each deletion are indicated above and below, respectively. These in frame deletions lead to the loss of S (622) and VSQD (1494-1497), respectively. (B) GBF1 is expressed at similar levels in wild-type CHO and mutant BFY-1 cell lines. Identical amounts of Triton X-100 extracts (30 μg) from the indicated cell lines were analyzed by PAGE/immunoblotting as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 and anti-G6PDH antibodies. The ratios of GBF1 to G6PDH were identical in both extracts.

Mentions: To determine whether GBF1 recovered from the BFY-1 library was a mutant allele, we isolated cDNAs from a library prepared from the wild-type parental CHO line by colony hybridization. Sequencing revealed that full length cDNAs recovered from the wild-type library had sequences identical to GBF1. Deletions of 3 and 12 nucleotides were observed at positions 1,864 and 4,479, respectively (Fig. 7 A), and reverse transcriptase–PCR analysis of mRNA prepared from both BFY-1 and parental CHO cells established that transcripts with and without those deletions were present at identical frequencies in both mutant and wild-type lines (not shown). Those probably arose by alternative processing. Furthermore, cDNAs identical in sequence to that shown in Fig. 7 A were recovered by colony hybridization of a twice selected BFY-1 cDNA library. The fact that GBF1 transcripts with identical sequences were recovered from wild-type and BFY-1 cells indicate that wild-type GBF1 is naturally BFA resistant. As expected, transfection of the wild-type cDNA diagrammed in Fig. 7 A led to recovery of BFA-resistant transformants (Table ). The human orthologue of GBF1 (Mansour et al., 1998) similarly caused BFA resistance when overexpressed (Melançon, P., unpublished observations).


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 is a naturally BFA-resistant GEF that is not overexpressed in BFY-1 cells. (A) Schematic representation of the GBF1 allele identified in wild-type CHO cells. The position of the Sec7d is indicated by the dark rectangle and the deletions by thin vertical lines. The size of the deletions and the position of the last nucleotide before each deletion are indicated above and below, respectively. These in frame deletions lead to the loss of S (622) and VSQD (1494-1497), respectively. (B) GBF1 is expressed at similar levels in wild-type CHO and mutant BFY-1 cell lines. Identical amounts of Triton X-100 extracts (30 μg) from the indicated cell lines were analyzed by PAGE/immunoblotting as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 and anti-G6PDH antibodies. The ratios of GBF1 to G6PDH were identical in both extracts.
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Related In: Results  -  Collection

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Figure 7: GBF1 is a naturally BFA-resistant GEF that is not overexpressed in BFY-1 cells. (A) Schematic representation of the GBF1 allele identified in wild-type CHO cells. The position of the Sec7d is indicated by the dark rectangle and the deletions by thin vertical lines. The size of the deletions and the position of the last nucleotide before each deletion are indicated above and below, respectively. These in frame deletions lead to the loss of S (622) and VSQD (1494-1497), respectively. (B) GBF1 is expressed at similar levels in wild-type CHO and mutant BFY-1 cell lines. Identical amounts of Triton X-100 extracts (30 μg) from the indicated cell lines were analyzed by PAGE/immunoblotting as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 and anti-G6PDH antibodies. The ratios of GBF1 to G6PDH were identical in both extracts.
Mentions: To determine whether GBF1 recovered from the BFY-1 library was a mutant allele, we isolated cDNAs from a library prepared from the wild-type parental CHO line by colony hybridization. Sequencing revealed that full length cDNAs recovered from the wild-type library had sequences identical to GBF1. Deletions of 3 and 12 nucleotides were observed at positions 1,864 and 4,479, respectively (Fig. 7 A), and reverse transcriptase–PCR analysis of mRNA prepared from both BFY-1 and parental CHO cells established that transcripts with and without those deletions were present at identical frequencies in both mutant and wild-type lines (not shown). Those probably arose by alternative processing. Furthermore, cDNAs identical in sequence to that shown in Fig. 7 A were recovered by colony hybridization of a twice selected BFY-1 cDNA library. The fact that GBF1 transcripts with identical sequences were recovered from wild-type and BFY-1 cells indicate that wild-type GBF1 is naturally BFA resistant. As expected, transfection of the wild-type cDNA diagrammed in Fig. 7 A led to recovery of BFA-resistant transformants (Table ). The human orthologue of GBF1 (Mansour et al., 1998) similarly caused BFA resistance when overexpressed (Melançon, P., unpublished observations).

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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