The Small GTPase Arf6 Functions as a Membrane Tether in a Chemically-Defined Reconstitution System.
Arf-family small GTPases are essential protein components for membrane trafficking in all eukaryotic endomembrane systems, particularly during the formation of membrane-bound, coat protein complex-coated transport carriers. In addition to their roles in the transport carrier formation, a number of Arf-family GTPases have been reported to physically associate with coiled-coil tethering proteins and multisubunit tethering complexes, which are responsible for membrane tethering, a process of the initial contact between transport carriers and their target subcellular compartments. Nevertheless, whether and how indeed Arf GTPases are involved in the tethering process remain unclear. Here, using a chemically-defined reconstitution approach with purified proteins of two representative Arf isoforms in humans (Arf1, Arf6) and synthetic liposomes for model membranes, we discovered that Arf6 can function as a bona fide membrane tether, directly and physically linking two distinct lipid bilayers even in the absence of any other tethering factors, whereas Arf1 retained little potency to trigger membrane tethering under the current experimental conditions. Arf6-mediated membrane tethering reactions require trans-assembly of membrane-anchored Arf6 proteins and can be reversibly controlled by the membrane attachment and detachment cycle of Arf6. The intrinsic membrane tethering activity of Arf6 was further found to be significantly inhibited by the presence of membrane-anchored Arf1, suggesting that the tethering-competent Arf6-Arf6 assembly in trans can be prevented by the heterotypic Arf1-Arf6 association in a cis configuration. Taken together, these findings lead us to postulate that self-assemblies of Arf-family small GTPases on lipid bilayers contribute to driving and regulating the tethering events of intracellular membrane trafficking.
['Fujibayashi|Kana|K|', 'Mima|Joji|J|']
small GTPase Arf6 membrane tethering reconstitution membrane trafficking Arf GTPase liposome