Characterization of the smallArabidopsis thalianaGTPase and ADP-ribosylation factor-like 2 protein TITAN5

Abstract

AbstractSmall 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) fromArabidopsis thaliana. TTN5 showed rapid guanine nucleotide exchange capacity comparable to that of human counterparts, but a remarkably low GTP hydrolysis reaction. A TTN5Q70Lmutant had enhanced nucleotide exchange activity, indicative of intracellular activation, while TTN5T30Nwith 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 andNicotiana benthamianaleaves. While YFP-TTN5 and YFP-TTN5Q70Lwere highly mobile in the cells, mobility was reduced for TTN5T30N. Colocalization with endomembrane markers in combination with pharmacological treatments resolved localization at membrane sites and showed that YFP-TTN5 and YFP-TTN5Q70Lwere located in Golgi stacks, multivesicular bodies, while this was less the case for YFP-TTN5T30N. 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.HighlightsThe small ARF-like GTPase TTN5 has a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanismBiochemical data classified TTN5 as a non-classical small GTPase, likely present in GTP-loaded active form in the cellYFP-TTN5 is dynamically associated with vesicle transport and different processes of the endomembrane system, requiring the active form of TTN5