Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN5

Small GTPases comprise key proteins in signal transduction that function by conformational switching ability between GDP- and GTP-bound states. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking and cellular functions. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized functional properties and cellular localization of the essential small ARF-like GTPase TITAN5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. TTN5 showed rapid guanine nucleotide exchange capacity comparable to that of human counterparts, but a remarkably low GTP hydrolysis reaction. A TTN5(Q70L) mutant had enhanced nucleotide exchange activity, indicative of intracellular activation, while TTN5(T30N) with fast nucleotide dissociation can be considered a dominant-negative form. This suggests that TTN5 is present in GTP-loaded active form in the cells. YFP-tagged TTN5 and the two derived mutant variants were located at multiple sites of the endomembrane system in the epidermis of Arabidopsis seedlings and Nicotiana benthamiana leaves. While YFP-TTN5 and YFP-TTN5(Q70L) were highly mobile in the cells, mobility was reduced for TTN5(T30N). Colocalization with endomembrane markers in combination with pharmacological treatments resolved localization at membrane sites and showed that YFP-TTN5 and YFP-TTN5(Q70L) were located in Golgi stacks, multivesicular bodies, while this was less the case for YFP-TTN5(T30N). On the other hand, all three TTN5 forms were located at the plasma membrane. Hence, the unusual capacity of rapid nucleotide exchange activity of the small ARF-like GTPase TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system.