Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

Abstract

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5' and 3' rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (approximately 208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a approximately 7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Figure 1

Identity of amino acid sequences of Sec7 (10) and the 200-kDa protein. An optimized alignment of the amino acid sequence of Yeast Sec7 and p200 is shown diagramatically. Above and below are numbers of amino acid positions in each protein. Percentage identity of amino acids in the boxed area is shown. The asterisk indicates the highly acidic region of Sec7.

Figure 2

Amino acid sequences of Sec7 domains of five ARF GEPs. The sequence of p200 is aligned with Gea1, cytohesin 1 (cyto), ARNO, and Sec7. Boxed residues are identical. Conservative differences are shaded. Consensus residues are identical in three or more sequences. Hyphens denote gaps introduced to maximize identity. Numbers in parentheses refer to position of the first amino acid in each Sec7 domain.

Figure 3

Northern blot analysis of p200 mRNA in bovine tissues. The blot with poly(A)⁺ RNA (2 μg) from the indicated bovine tissues was hybridized with a 1.3-kb cDNA representing nucleotides 43–1,329 from p200 and then after stripping, with β-actin cDNA. Positions of size markers (kb) are indicated.

Figure 4

(A) Purification of p200 synthesized as a six-His fusion protein in Sf9 cells. After separation by SDS/PAGE in 10% gel, proteins were stained with Coomassie blue. Lanes: 1, soluble proteins (30 μg) from lysate of induced Sf9 cells; 2, affinity-purified six-His-tagged p200 (≈100 ng); 3, standard proteins, size (kDa) on the right. (B) Effect of recombinant six-His-tagged p200 on ARF activity. ARF activity is CTA activity (nmol of ADP-ribosylagmatine synthesized per hr) with ARF minus that without ARF. Assays with the indicated amounts of purified six-His-tagged-p200 (≈3 μg/ml) were carried out without and with 6 μg of BFA. Data are means ± SEM of values of two samples. This experiment was replicated three times with different preparations. Activity of the partially purified ARF (10 μg) was equivalent to ≈0.1 μg of purified ARF3. (C) Effect of BFA on six-His-tagged p200 stimulation of GTPγS binding by ARF. Assays with the indicated amount of purified six-His-tagged-p200 (≈3 μg/ml) and 10 μg of partially purified native ARFs without or with 6 μg of BFA were incubated for 20 min at 30°C. Data are means ± SEM of values from triplicate assays. This experiment was replicated twice.