Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress

Abstract

A cDNA encoding a member of the Ypt/Rab family of small GTP-binding proteins was cloned from the facultative CAM plant Mesembryanthemum crystallinum. Mcrab5b includes an open reading frame of 201 amino acids. The deduced amino acid sequence shows 91% similarity to LjRAB5b isolated from Lotus japonicus. The amino acid sequence of McRAB5b provides interesting features suggesting that McRAB5b and its homologue from Lotus japonicus represent a new subclass of Ypt/Rab proteins. The fact that proteins like McRAB5b and LjRAB5b were only found in plants and not in yeast or vertebrates suggests that they have plant-specific functions. The expression of Mcrab5b as investigated by northern blot hybridization and RT-PCR was stimulated under salt stress. After heterologous expression in Escherichia coli an antibody was raised against recombinant McRABSb protein. Western blot analysis revealed that McRAB5b was bound to membranes. It is present in a monomeric and a dimeric form in vitro and in vivo. In vitro only the monomeric protein exhibits a binding capacity for radiolabelled GTP, while the dimer is unable to do so, indicating that the activity may be regulated by monomer/dimer transition.