Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN5
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
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.
Highlights
-
The small ARF-like GTPase TTN5 has a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism
-
Biochemical data classified TTN5 as a non-classical small GTPase, likely present in GTP-loaded active form in the cell
-
YFP-TTN5 is dynamically associated with vesicle transport and different processes of the endomembrane system, requiring the active form of TTN5