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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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ARF-GEF and COPI recruitment activity on Golgi membranes from cells expressing GBF1 is resistant to BFA. (A) Identical amounts (4.2 μg) of Golgi-enriched membrane fractions from 293 cells or 293 cells transformed with GBF1 were assayed for ARF nucleotide exchange in the presence of α[32P]GTP and in the absence or presence of BFA (280 μM). Each bar represents the average of three determinations ± SD. Similar results were obtained with several independently obtained membrane preparations. (B) Coatomer recruitment assays contained Golgi membranes prepared from 293 cells transformed with either pCEP4 (open circle) or pCEP4-GBF1 (closed circle) and the indicated amounts of BFA. The extent of recruitment of COPI provided by BFA-sensitive cytosol was determined in triplicate as described in Materials and Methods. The results ± SD are plotted as a function of BFA concentration. Similar results were obtained with several different membrane preparations.
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Figure 4: ARF-GEF and COPI recruitment activity on Golgi membranes from cells expressing GBF1 is resistant to BFA. (A) Identical amounts (4.2 μg) of Golgi-enriched membrane fractions from 293 cells or 293 cells transformed with GBF1 were assayed for ARF nucleotide exchange in the presence of α[32P]GTP and in the absence or presence of BFA (280 μM). Each bar represents the average of three determinations ± SD. Similar results were obtained with several independently obtained membrane preparations. (B) Coatomer recruitment assays contained Golgi membranes prepared from 293 cells transformed with either pCEP4 (open circle) or pCEP4-GBF1 (closed circle) and the indicated amounts of BFA. The extent of recruitment of COPI provided by BFA-sensitive cytosol was determined in triplicate as described in Materials and Methods. The results ± SD are plotted as a function of BFA concentration. Similar results were obtained with several different membrane preparations.

Mentions: It has been previously reported that BFA inhibits ARF activation and coat recruitment both in vivo and in vitro, indicating that BFA acts at or upstream of the ARF guanine nucleotide exchange activity (Donaldson et al. 1992b; Helms and Rothman 1992; Randazzo et al. 1993). To test whether GBF1 acts at this level, we used cell-free assays that measure membrane-associated ARF-GEF activity and recruitment of COPI. Golgi-enriched membrane fractions were prepared from either control or GBF1-expressing 293 cells. GEF assays performed with native ARFs obtained from bovine brain (predominantly ARF3) established that Golgi-enriched membrane fractions prepared from 293/GBF1 cells displayed normal levels of ARF-GEF activity (Fig. 4 A). However, in contrast to that observed with control membranes, this activity was completely resistant to BFA. To establish whether the BFA-resistant ARF-GEF activity was relevant to coatomer recruitment, we compared the ability of 293 and 293/GBF1 membranes to recruit COPI components. As observed with the nucleotide exchange assay, 293/GBF1 membranes recruited levels of COPI nearly identical to those measured with control membranes (Fig. 4 B). Furthermore, COPI recruitment on these membranes was barely affected at a BFA concentration as high as 70 μM when recruitment to control membranes was inhibited by >50% at 7 μM BFA (Fig. 4 B).


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)

ARF-GEF and COPI recruitment activity on Golgi membranes from cells expressing GBF1 is resistant to BFA. (A) Identical amounts (4.2 μg) of Golgi-enriched membrane fractions from 293 cells or 293 cells transformed with GBF1 were assayed for ARF nucleotide exchange in the presence of α[32P]GTP and in the absence or presence of BFA (280 μM). Each bar represents the average of three determinations ± SD. Similar results were obtained with several independently obtained membrane preparations. (B) Coatomer recruitment assays contained Golgi membranes prepared from 293 cells transformed with either pCEP4 (open circle) or pCEP4-GBF1 (closed circle) and the indicated amounts of BFA. The extent of recruitment of COPI provided by BFA-sensitive cytosol was determined in triplicate as described in Materials and Methods. The results ± SD are plotted as a function of BFA concentration. Similar results were obtained with several different membrane preparations.
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Related In: Results  -  Collection

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Figure 4: ARF-GEF and COPI recruitment activity on Golgi membranes from cells expressing GBF1 is resistant to BFA. (A) Identical amounts (4.2 μg) of Golgi-enriched membrane fractions from 293 cells or 293 cells transformed with GBF1 were assayed for ARF nucleotide exchange in the presence of α[32P]GTP and in the absence or presence of BFA (280 μM). Each bar represents the average of three determinations ± SD. Similar results were obtained with several independently obtained membrane preparations. (B) Coatomer recruitment assays contained Golgi membranes prepared from 293 cells transformed with either pCEP4 (open circle) or pCEP4-GBF1 (closed circle) and the indicated amounts of BFA. The extent of recruitment of COPI provided by BFA-sensitive cytosol was determined in triplicate as described in Materials and Methods. The results ± SD are plotted as a function of BFA concentration. Similar results were obtained with several different membrane preparations.
Mentions: It has been previously reported that BFA inhibits ARF activation and coat recruitment both in vivo and in vitro, indicating that BFA acts at or upstream of the ARF guanine nucleotide exchange activity (Donaldson et al. 1992b; Helms and Rothman 1992; Randazzo et al. 1993). To test whether GBF1 acts at this level, we used cell-free assays that measure membrane-associated ARF-GEF activity and recruitment of COPI. Golgi-enriched membrane fractions were prepared from either control or GBF1-expressing 293 cells. GEF assays performed with native ARFs obtained from bovine brain (predominantly ARF3) established that Golgi-enriched membrane fractions prepared from 293/GBF1 cells displayed normal levels of ARF-GEF activity (Fig. 4 A). However, in contrast to that observed with control membranes, this activity was completely resistant to BFA. To establish whether the BFA-resistant ARF-GEF activity was relevant to coatomer recruitment, we compared the ability of 293 and 293/GBF1 membranes to recruit COPI components. As observed with the nucleotide exchange assay, 293/GBF1 membranes recruited levels of COPI nearly identical to those measured with control membranes (Fig. 4 B). Furthermore, COPI recruitment on these membranes was barely affected at a BFA concentration as high as 70 μM when recruitment to control membranes was inhibited by >50% at 7 μM BFA (Fig. 4 B).

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