The presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1

Abstract

Two new members (Bsar1a and Bsar1b) of the Sari gene family have been identified from a flower bud cDNA library of Brassica campestris and their functional characteristics were analyzed. The two clones differ from each other at 14 positions of the 193 amino acid residues deduced from their coding region. The amino acid sequences of Bsar1a and Bsar1b are most closely related to the Sari family, genes that function early in the process of vesicle budding from the endoplasmic reticulum (ER). The sequences contain all the conserved motifs of the Rns superfamily (G1-G4 motifs) as well as the distinctive structural feature near the C-terminus that is Sari specific. Our phylogenetic analysis confirmed that these two clones can indeed be considered members of the Sari family and that they have a close relationship to the ARF family. The Bsar1 proteins, expressed in Escherichia coli, cross-reacted with a polyclonal antibody prepared against Saccharomyces cerevisiae Sari protein. It also exhibited GTP-binding activity. Genomic Southern blot analysis, using the 3'-gene-specific regions of the Bsar1 cDNAs as probes, revealed that the two cDNA clones are members of a B. campestris Sari family that consists of 2 to 3 genes. RNA blot analysis, using the same gene-specific probes, showed that both genes are expressed with similar patterns in most tissues of the plant, including leaf, stem, root, and flower buds. Furthermore, when we placed the two Bsar1 genes under the control of the yeast pGK1 promoter into the temperature-sensitive mutant yeast strain S. cerevisiae Sec12-1, they suppressed the mutation which consists of a defect in vesicle transport. The amino acid sequence similarity, the GTP-binding activity, and the functional suppression of the yeast mutation suggest that the Bsar1 proteins are functional homologues of the Sari protein in S. cerevisiae and that they may perform similar biological functions.