GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5

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.

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.

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.

Figure 3

The Golgi morphology of 293 overexpressing GBF1 is resistant to BFA. 293 cells were transformed as described with pCEP4 vector (control) or pCEP4-GBF1 (GBF1) and grown on coverslips. The cells were incubated in the absence (A and B) or in the presence of 4 μM BFA (C and D) for 20 min before fixation and staining with antigiantin serum as described in Materials and Methods. Bar, 10 μm.

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.

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.

Figure 6

(His)₆-GBF1 is a BFA-resistant ARF-GEF. (A) Fractions enriched in (His)₆-GBF1 display a GEF specific for ARFs. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (vector alone) and (His)₆GBF1 extracts were assayed for loading of small GTPases with GTPγS in 1 μM of free Mg²⁺ as described in Materials and Methods. Purified ARF1/3, Sar1p, or rab1b (1 μM each) were used as substrate as indicated. Each bar represents the average of three determinations ± SD. (B) GEF activity of GBF1 towards ARF1/3 is BFA resistant. Time course of ARF1/3 nucleotide exchange using the 50 mM imidazole eluate fraction from the (His)₆GBF1 extracts. Assays were performed at 1 μM of free Mg²⁺ either in the presence (closed circle) or absence (open circle) of BFA (360 μM). Similar results were obtained with several independent preparations. (C) The purified Sec7 domain of Sec7 is a BFA-sensitive ARF-GEF. Assays were performed as described in Materials and Methods with the indicated amounts of BFA. Each bar represents the average of three determinations ± SD. (D) GBF1 is a BFA-resistant GEF specific for ARF5 at 1 mM Mg²⁺. Identical volumes (5 μl) of the 50 mM imidazole eluate fractions from the control (empty vector) and (His)₆GBF1 extracts were assayed for loading of ARF1/3 or ARF5 in assays containing 1 mM Mg²⁺. BFA was used at a concentration of 600 μM. One set of reactions was performed with the GBF1 fraction inactivated (asterisk) by incubation at 95°C for 5 min. Each bar represents the average of three determinations ± SD.

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.

Figure 9

GBF1 localizes primarily to a tubular network proximal to Golgi stacks. Rat liver frozen cryosections were processed, labeled, and photographed as described in Materials and Methods. (A) anti-GBF1 serum H134 (diluted 1:2). (B) anti-GBF1 serum H133 (diluted 1:5). (C and D) mouse anti–β-COP (diluted 1:20). (Arrowheads) Gold particles labeling ER cisternae. (Arrows) Gold particles labeling tubular networks proximal to the Golgi complex. (Curved arrows) Gold particles labeling Golgi stacks. (Brackets) High concentration of gold particles labeling tubular networks proximal to the Golgi complex. M, mitochondria; P, peroxisome; and G, Golgi stack. Bars, 400 nm.

Figure 8

GBF1 colocalizes with β-COP to the Golgi complex. (A) Wild-type CHO (left), mutant BFY-1 (center), and NRK (right) cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and stained with a Texas red–conjugated secondary antibody. Images obtained by standard epifluorescence microscopy are presented. (B) NRK cells were permeabilized and incubated with rabbit serum H154 (anti-GBF1) and mAb m3A5 (anti–β-COP) and stained with FITC- (β-COP) and Texas red– (GBF1) conjugated secondary antibodies. The images presented were obtained by confocal microscopy. (Left) GBF1 signal; (right) β-COP signal; and (center) merge of both images. Bars, 10 μm.