FUNCTIONAL COMPLEMENTATION OF A YEAST VESICULAR TRANSPORT MUTATION YPT1-1 BY A BRASSICA-NAPUS CDNA CLONE ENCODING A SMALL GTP-BINDING PROTEIN

Abstract

A cDNA clone (bra) encoding a small GTP-binding protein was isolated from Brassica napus by screening a root cDNA library with a degenerate oligonucleotide probe that corresponds to a highly conserved GTP-binding domain of the Pas superfamily. Sequence analysis shows that the clone contains an open reading frame of 219 amino acid residues with the estimated molecular mass of 24 379 Da and this coding region contains all the conserved motifs of the Pas superfamily. The deduced amino acid sequence of the bra gene is most closely related to the Ypt/Rab family that functions in the vesicular transport (46% and 47% amino acid identity to the yeast Ypt1 and to the human Rab1, respectively) and is more distantly related to the other Pas-related families. The protein encoded by the bra gene, when expressed in Escherichia coli, shows the ability to bind GTP. Furthermore, when the bra gene is introduced into Saccharomyces cerevisiae under the regulation of the yeast GAL1 promoter, the gene can complement the temperature-sensitive yeast mutation ypt1-1 that has defects in vesicular transport function. The amino acid sequence similarity and the functional complementation of the yeast mutation suggest that this gene is likely to be involved in the vesicular transport in plants. Genomic Southern analysis shows that this gene is a member of a small gene family in Brassica napus.