A specific feature of the angiosperm Rab escort protein (REP) and evolution of the REP/GDI superfamily.
Rab GTPases participating in the regulation of vesicle trafficking in eukaryotes are geranylgeranylated by the Rab geranylgeranyl transferase (RabGGTase) in complex with the Rab escort protein (REP). Here, we describe basic properties of the Arabidopsis thaliana REP (AthREP), first REP outside yeasts or metazoans to be characterized. GFP-tagged AthREP, as well as the geranylgeranylation activity, were localized predominantly to the cytoplasm. Recombinant AthREP interacted with yeast 6His-Ypt1, tobacco 6His-RabA1a, and Arabidopsis RabA2a in vitro preferring the GDP-bound form of the latter. Recombinant AthREP with C-terminal but not N-terminal tags stimulated geranylgeranylation of various Rab GTPases in Arabidopsis extracts in vitro. Neither recombinant AthREP protein exhibited activity in yeast extracts, while recombinant yeast REP (6His-SceMrs6) stimulated Rab geranylgeranylation in all extracts tested. We found that a conserved arginine residue, R195, known to be crucial for yeast REP function, is substituted by an asparagine or threonine residue in angiosperm REPs. A point mutant allele of AthREP with arginine at this position complemented the yeast REP mutation, while wild-type AthREP did not. Based on phylogenetic analysis of REP and GDP dissociation inhibitor (GDI) sequences from a broad range of eukaryotic lineages, we propose a new view on evolution of the REP/GDI superfamily with a bi-functional REP/GDI protein as a direct ancestor.
Hála
Michal
M
Institute of Experimental Botany, Rozvojová 135, Praha 6-Lysolaje, 165 02, Czech Republic.
Eliás
Marek
M
Zárský
Viktor
V
D048868:Adaptor Proteins, Signal Transducing; D019883:Alkyl and Aryl Transferases / Q000145:classification* / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry; D019143:Evolution, Molecular; D005816:Genetic Complementation Test; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D017354:Point Mutation / Q000235:genetics; D017368:Protein Prenylation / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D029681:Arabidopsis Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; C092439:MRS6 protein, S cerevisiae; C513165:Rab escort protein, Arabidopsis; D029701:Saccharomyces cerevisiae Proteins; D019883:Alkyl and Aryl Transferases; D020691:rab GTP-Binding Proteins