Mutational analysis of activation mechanism of Arabidopsis Rab5 group

Abstract

Rab5 GTPase is known to regulate not only the endosomal fusion but also signaling through endosomes.The Rab GTPases are activated by specific guanine nucleotide exchange factors (GEFs), which accelerates the exchange of GDP to GTP. The Vps9 domain, the catalytic core for the activation of Rab5, is conserved in all Rab5 GEFs. We have already demonstrated that the AtVps9a can activate all Arabidopsis Rab5 members (Ara6,Ara7 and Rha1). To examine the activation mechanism of Rab5s in detail, we performed mutational analysis of AtVps9a based on the crystal structure of AtVps9a/Ara7 complex. We introduced several amino acid changes in Vps9 domain and revealed that conserved Tyr (225) and Asp (185) in the Vps9 domain are essential for the GEF activity. We also demonstrated that truncated AtVps9a shows specific increase in the GEF activity toward Ara6. We are now examining the effect of these mutations on plant development using transgenic Arabidopsis.