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A cDNA Encoding a New GTP-Binding Protein of the ADP-Ribosylation Factor Family from Arabidopsis

The low mo1 wt GTP-binding protein superfamily ras includes ARFs. Their function depends on their ability to altemate between active and inactive states by association to GTP and GDP, respectively. The first ARF protein was identified in mammalian cells as a cofactor required for ADPribosylation of the adenylate cyclase G,, subunit catalyzed by cholera toxin in vitro (Kahn and Gilman, 1984). Widespread expression of the highly conserved ARF proteins in eukaryotic cells raised the question of their physiological role in cell regulation. Recent studies have shown that these proteins were involved in various vesicular traffic steps of the exocytic and endocytic pathways (Serafini et al., 1991; Balch et al., 1992; Kahn et al., 1992; Lenhard et al., 1992) and in nuclear envelope assembly (Boman et al., 1992). In higher plants, ARFs were first described when we reported the isolation of a cDNA coding for an ARF homolog (ARF1) from Arabidopsis (Regad et al., 1993; accession No. M95166). Recently, an ARF-type protein has been detected in Pisum sativum (Memon et al., 1993). On the other hand, genes of an ARF subfamily coding for structurally related proteins were characterized in Drosophila and human genomic libraries (Tamkun et al., 1991; Clark et al., 1993). These ARLs are functionally distinct from ARFs by virtue of their complete lack of in vitro ADP-ribosylation activity. By sequencing of randomly selected cDNA clones from an Arabidopsis cultured cell library, we identified a clone (Atadprf3) encoding a new ARF protein homolog (ARF3) that is different from the previously reported Arabidopsis ARFl protein (Table I). Both nucleotideand DNA-deduced amino acid sequences of ARF3 exhibit a somewhat low homology with those of ARFl, corresponding to 62 and 61% identity, respectively. The Arabidopsis ARF3 protein has been compared to the 27 DNA-deduced amino acid sequences of the ARF family available to date in data bases. Surprisingly, ARF3 is closest to the Drosophila ARL called ar1 characterized by Tamkun et al. (1991). ARF3 and ar1 are 64% identical, their homology reachmg 74% for the 100 amino acids of the amino-terminal region. This suggests that Arabidopsis ARF3 may belong to the ARL protein class. However, ARF3 is