Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN 5.

Small GTPases switch between GDP- and GTP-bound states in cell signaling. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. We characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5/HALLIMASCH/ARL2/ARLC1 from Arabidopsis thaliana, including two conserved point mutants, suspected to be functional for nucleotide exchange and GTP hydrolysis, TTN5T30N and TTN5Q70L. TTN5 exhibited very rapid intrinsic nucleotide exchange and remarkably low GTP hydrolysis activity, functioning as a non-classical small GTPase being likely present in a GTP-loaded active form. We analyzed signals from YFP-TTN5 and HA3-TTN5 in situ immunolocalization in Arabidopsis seedlings and in transient expression system. Colocalization with endomembrane markers and pharmacological treatments suggests that TTN5 can be present at the plasma membrane and dynamically associated with membranes of vesicles, Golgi stacks and multivesicular bodies. While TTN5Q70L mirrored wild-type TTN5 behavior, TTN5T30N mutant differed in some aspects. Hence, the unusual rapid nucleotide exchange activity of TTN5 is linked with membrane dynamics, likely associated with vesicle transport in the endomembrane system.