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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 anti–C-tail peptide antibody detects a 206-kD protein consistent with the predicted size of GBF1. (A) Serum H154 recognizes a 206-kD protein that is expressed at normal levels in mutant BFY-1 cells but is overexpressed in 293 cells transfected with pCEP4-GBF1. 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 the indicated sera and processed for enhanced chemifluorescence. Scans obtained with a PhosphorImager in fluorescence mode are shown. The size of molecular weight standards run in parallel are indicated. (B) GBF1 is primarily cytosolic when overexpressed in 293/GBF1 cells. Identical amount (90 μg) of postnuclear supernatants from the indicated cells were analyzed by PAGE/immunoblotting either directly or after separation into cytosol and membrane fractions by ultracentrifugation as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 serum and anticalnexin antibodies before processing for enhanced chemifluorescence. The distribution of the membrane protein calnexin confirms the absence of microsomes in the cytosol and their recovery in the pellet fraction. (C) GBF1 is primarily cytosolic in BFY-1 cells. The fractionation and detection were as described for B.
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Figure 5: The anti–C-tail peptide antibody detects a 206-kD protein consistent with the predicted size of GBF1. (A) Serum H154 recognizes a 206-kD protein that is expressed at normal levels in mutant BFY-1 cells but is overexpressed in 293 cells transfected with pCEP4-GBF1. 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 the indicated sera and processed for enhanced chemifluorescence. Scans obtained with a PhosphorImager in fluorescence mode are shown. The size of molecular weight standards run in parallel are indicated. (B) GBF1 is primarily cytosolic when overexpressed in 293/GBF1 cells. Identical amount (90 μg) of postnuclear supernatants from the indicated cells were analyzed by PAGE/immunoblotting either directly or after separation into cytosol and membrane fractions by ultracentrifugation as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 serum and anticalnexin antibodies before processing for enhanced chemifluorescence. The distribution of the membrane protein calnexin confirms the absence of microsomes in the cytosol and their recovery in the pellet fraction. (C) GBF1 is primarily cytosolic in BFY-1 cells. The fractionation and detection were as described for B.

Mentions: To measure the extent of GBF1 overexpression and assess its distribution, we prepared and characterized several antisera raised against a peptide corresponding to the COOH terminus of GBF1 (see Materials and Methods). These antisera recognized specifically a protein of 206 kD in both CHO and 293 cells, a size similar to that predicted from the sequence of the cDNA (Fig. 5 A and data not shown). BFA-resistant 293 transformants overexpressed GBF1 six- to eightfold above the endogenous protein level (Fig. 5 A, right). As predicted, the majority of overexpressed GBF1 in 293/GBF1 cells was recovered in cytosolic extracts under conditions where microsomes were efficiently removed as established with the membrane protein calnexin (Fig. 5 B). Endogenous GBF1 in BFY-1 cells also partitioned primarily to the cytosol (Fig. 5 C); quantitation of this and similar experiments established that only a small fraction (< 10%) of endogenous protein was recovered in the microsome pellet. Similar results were obtained for the endogenous protein in wild-type 293 and CHO cells (not shown). Our observations suggest that GBF1 is a primarily soluble protein implicated in coatomer recruitment.


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 anti–C-tail peptide antibody detects a 206-kD protein consistent with the predicted size of GBF1. (A) Serum H154 recognizes a 206-kD protein that is expressed at normal levels in mutant BFY-1 cells but is overexpressed in 293 cells transfected with pCEP4-GBF1. 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 the indicated sera and processed for enhanced chemifluorescence. Scans obtained with a PhosphorImager in fluorescence mode are shown. The size of molecular weight standards run in parallel are indicated. (B) GBF1 is primarily cytosolic when overexpressed in 293/GBF1 cells. Identical amount (90 μg) of postnuclear supernatants from the indicated cells were analyzed by PAGE/immunoblotting either directly or after separation into cytosol and membrane fractions by ultracentrifugation as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 serum and anticalnexin antibodies before processing for enhanced chemifluorescence. The distribution of the membrane protein calnexin confirms the absence of microsomes in the cytosol and their recovery in the pellet fraction. (C) GBF1 is primarily cytosolic in BFY-1 cells. The fractionation and detection were as described for B.
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Related In: Results  -  Collection

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Figure 5: The anti–C-tail peptide antibody detects a 206-kD protein consistent with the predicted size of GBF1. (A) Serum H154 recognizes a 206-kD protein that is expressed at normal levels in mutant BFY-1 cells but is overexpressed in 293 cells transfected with pCEP4-GBF1. 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 the indicated sera and processed for enhanced chemifluorescence. Scans obtained with a PhosphorImager in fluorescence mode are shown. The size of molecular weight standards run in parallel are indicated. (B) GBF1 is primarily cytosolic when overexpressed in 293/GBF1 cells. Identical amount (90 μg) of postnuclear supernatants from the indicated cells were analyzed by PAGE/immunoblotting either directly or after separation into cytosol and membrane fractions by ultracentrifugation as described in Materials and Methods. Blots were incubated with H154 anti-GBF1 serum and anticalnexin antibodies before processing for enhanced chemifluorescence. The distribution of the membrane protein calnexin confirms the absence of microsomes in the cytosol and their recovery in the pellet fraction. (C) GBF1 is primarily cytosolic in BFY-1 cells. The fractionation and detection were as described for B.
Mentions: To measure the extent of GBF1 overexpression and assess its distribution, we prepared and characterized several antisera raised against a peptide corresponding to the COOH terminus of GBF1 (see Materials and Methods). These antisera recognized specifically a protein of 206 kD in both CHO and 293 cells, a size similar to that predicted from the sequence of the cDNA (Fig. 5 A and data not shown). BFA-resistant 293 transformants overexpressed GBF1 six- to eightfold above the endogenous protein level (Fig. 5 A, right). As predicted, the majority of overexpressed GBF1 in 293/GBF1 cells was recovered in cytosolic extracts under conditions where microsomes were efficiently removed as established with the membrane protein calnexin (Fig. 5 B). Endogenous GBF1 in BFY-1 cells also partitioned primarily to the cytosol (Fig. 5 C); quantitation of this and similar experiments established that only a small fraction (< 10%) of endogenous protein was recovered in the microsome pellet. Similar results were obtained for the endogenous protein in wild-type 293 and CHO cells (not shown). Our observations suggest that GBF1 is a primarily soluble protein implicated in coatomer recruitment.

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