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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 novel member of the Sec7 family of proteins. (A) Deduced amino acid sequence of GBF1. The Sec7 domain of this protein (170 amino acids) is indicated in bold characters. The two highly conserved motif 1 and motif 2 within this domain are surrounded by boxes. The sequence data are available from GenBank/EMBL/DDBJ under accession number AF127523. (B) Multiple alignment of GBF1 and other key members of the Sec7 family of proteins generated with the MACAW program. The name of each protein is indicated on the left of each peptide diagram. The number of amino acid residues is indicated on the right. Boxes indicate regions of significant homology among two or more proteins. Darker shading of homology boxes indicates increased representation of these domains across family members. Small members of the family are represented by the better characterized ARNO.
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Figure 2: GBF1 is a novel member of the Sec7 family of proteins. (A) Deduced amino acid sequence of GBF1. The Sec7 domain of this protein (170 amino acids) is indicated in bold characters. The two highly conserved motif 1 and motif 2 within this domain are surrounded by boxes. The sequence data are available from GenBank/EMBL/DDBJ under accession number AF127523. (B) Multiple alignment of GBF1 and other key members of the Sec7 family of proteins generated with the MACAW program. The name of each protein is indicated on the left of each peptide diagram. The number of amino acid residues is indicated on the right. Boxes indicate regions of significant homology among two or more proteins. Darker shading of homology boxes indicates increased representation of these domains across family members. Small members of the family are represented by the better characterized ARNO.

Mentions: Analysis of the predicted amino acid sequence (Fig. 2) revealed a novel 206-kD protein with significant homology to a family of proteins that contains a 170-residue domain, called Sec7 domain (Sec7d), first identified in the secretion protein Sec7p of S. cerevisiae. This homology is significant since many Sec7 domain–containing proteins have been implicated in ARF guanine nucleotide exchange (Chardin et al. 1996; Peyroche et al. 1996; Meacci et al. 1997). Furthermore, the Sec7d itself has been shown to possess an intrinsic ARF-GEF activity (Chardin et al. 1996; Morinaga et al. 1997; Sata et al. 1998; Mansour et al. 1999).


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 novel member of the Sec7 family of proteins. (A) Deduced amino acid sequence of GBF1. The Sec7 domain of this protein (170 amino acids) is indicated in bold characters. The two highly conserved motif 1 and motif 2 within this domain are surrounded by boxes. The sequence data are available from GenBank/EMBL/DDBJ under accession number AF127523. (B) Multiple alignment of GBF1 and other key members of the Sec7 family of proteins generated with the MACAW program. The name of each protein is indicated on the left of each peptide diagram. The number of amino acid residues is indicated on the right. Boxes indicate regions of significant homology among two or more proteins. Darker shading of homology boxes indicates increased representation of these domains across family members. Small members of the family are represented by the better characterized ARNO.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199737&req=5

Figure 2: GBF1 is a novel member of the Sec7 family of proteins. (A) Deduced amino acid sequence of GBF1. The Sec7 domain of this protein (170 amino acids) is indicated in bold characters. The two highly conserved motif 1 and motif 2 within this domain are surrounded by boxes. The sequence data are available from GenBank/EMBL/DDBJ under accession number AF127523. (B) Multiple alignment of GBF1 and other key members of the Sec7 family of proteins generated with the MACAW program. The name of each protein is indicated on the left of each peptide diagram. The number of amino acid residues is indicated on the right. Boxes indicate regions of significant homology among two or more proteins. Darker shading of homology boxes indicates increased representation of these domains across family members. Small members of the family are represented by the better characterized ARNO.
Mentions: Analysis of the predicted amino acid sequence (Fig. 2) revealed a novel 206-kD protein with significant homology to a family of proteins that contains a 170-residue domain, called Sec7 domain (Sec7d), first identified in the secretion protein Sec7p of S. cerevisiae. This homology is significant since many Sec7 domain–containing proteins have been implicated in ARF guanine nucleotide exchange (Chardin et al. 1996; Peyroche et al. 1996; Meacci et al. 1997). Furthermore, the Sec7d itself has been shown to possess an intrinsic ARF-GEF activity (Chardin et al. 1996; Morinaga et al. 1997; Sata et al. 1998; Mansour et al. 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.

Show MeSH
Related in: MedlinePlus