Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor

Abstract

Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport. More recently, the same protein has been reported to be a regulatory factor for the alphaLbeta2 integrin in lymphocytes. Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function. ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP). Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP. Both soluble and particulate GEPs have been described. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated. Cytohesin-1 enhanced binding of 35S-labeled guanosine 5'-[gamma-thio]triphosphate [35S]GTP[gammaS] or [3H]GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP). Addition of cytohesin-1 to ARF3 with [35S]GTP[gammaS] bound, accelerated [35S]GTP[gammaS] release to a similar degree in the presence of unlabeled GDP or GTP[gammaS] and to a lesser degree with GDP[betaS]; release was negligible without added nucleotide. Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function. In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.

Figure 1

Alignment of sequences of Sec7 domains in related proteins. Sec7 domains of cytohesin-1 (B2–1; amino acids 62–249), Sec7 (amino acids 827-1017), EMB30 (amino acids 557–752), and Caenorhabditis elegans cosmid K06H7 (K06H7) sequences were aligned using clustal (pc gene). The Sec7 domains of B2–1 and KO6H7 contain 188 amino acids; Sec7, 189; and EMB30, 196.

Figure 2

Effect of cytohesin-1 on [³⁵S]GTP[γS] binding to ARF3. Native ARF3 (0.25 μg, ≈12.5 pmol) was incubated with zero (□), 0.06 (•), 0.12 (▴), or 0.24 μg (▾) of cytohesin-1 and 4 μM [³⁵S]GTP[γS] (2 × 10⁶ cpm) in 100 μl containing 20 mM Tris (pH 8.0), 10 mM imidazole, 100 mM NaCl, 10 μg of phosphatidylserine, 40 μg of BSA, 10 mM DTT, and 3.1 mM MgCl₂ at 36.5°C for the time indicated, then at 0°C for a total of 60 min. Proteins were collected on nitrocellulose filters for radioassay of bound GTP[γS]. GTP[γS] bound to samples without ARF3 or cytohesin-1 after 60 min at 0°C was 0.1 pmol, to ARF3 was 0.14 pmol, and to cytohesin-1 alone was 0.11 pmol. After 60 min at 0°C, 12.5 pmol of ARF3 plus 0.06 μg, 0.12 μg, or 0.24 μg of cytohesin-1 bound 0.99, 1.2, and 1.53 pmol, respectively. Control values have been subtracted from the data presented.

Figure 3

Effect of cytohesin-1 on [³H]GDP binding to ARF3. Samples of ARF3 (15 pmol) were incubated with zero (□), 0.06 (•), or 0.2 μg (▴) of cytohesin-1; 2 μM [³H]GDP (7.5 × 10⁵ cpm); and 4 mM MgCl₂ in 100 μl of the medium described in Fig. 2, at 36°C for the indicated time before proteins with bound [³H]GDP were collected on nitrocellulose for radioassay. Samples without ARF3, with or without cytohesin-1, bound 0.06–0.08 pmol; these control values have been subtracted from the data presented.

Figure 4

Effect of cytohesin-1 on release of bound GTP[γS] from ARF3. Samples of native ARF3 (1.5 μg, 75 pmol) were incubated with 4 μM [³⁵S]GTP[γS] (2 × 10⁶ cpm), as described (12), in 100 μl containing 20 mM Tris (pH 8.0), 50 mM NaCl, 20 μg of phosphatidylserine, 50 μg of BSA, 12 mM DTT, 0.65 mM MgCl₂, and 1 mM EDTA for 40 min at 36.5°C. Tubes were placed in ice and 20-μl samples (15 pmol of ARF3) were transferred to tubes containing 50 μM unlabeled GTP[γS] (▪), GDP (•), GDP[βS] (▴), or no nucleotide (▾), with (solid line) or without (dotted line) cytohesin-1 (0.5 μg) and other additions, as indicated in Fig. 2, in a final volume of 100 μl. After incubation for the indicated time, samples were filtered for radioassay of protein-bound [³⁵S]GTP[γS]. One hundred percent binding (3.1–3.2 pmol) was the mean of values from samples of ARF3 ± cytohesin-1 and unlabeled nucleotide kept at 0°C for 60 min. Without ARF3, 0.03–0.05 pmol of [³⁵S]GTP[γS] was bound; these control values have been subtracted from the data presented.

Figure 5

Effect of nucleotide concentration on release of bound GTP[γS] from ARF3 with or without cytohesin-1. ARF3 (1.5 μg, 75 pmol) was incubated with 4 μM [³⁵S]GTP[γS], as described in Fig. 4, and cooled. Samples (20 μl) were then incubated with (▪, •) or without (□, ○) cytohesin-1 (0.5 μg), and the indicated concentration of GTP[γS] (○, •) or GDP (□, ▪) for 40 min at 36.5°C before assay of protein-bound [³⁵S]GTP[γS]. One hundred percent binding (3.1–3.2 pmol) was the mean of values for ARF3 ± cytohesin-1 and unlabeled nucleotide incubated at 0°C for 60 min. Control values (0.06–0.08 pmol bound in the absence of ARF3) have been subtracted.