Abstract
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.
Highlights
Small GTPases of the Arf (ADP-ribosylation factor) family, belonging to the Ras superfamily, are known to be essential protein components for intracellular membrane trafficking in all eukaryotic endomembrane systems and, in general, to function through cooperation with their specific interactors, termed Arf effectors (D’Souza-Schorey and Chavrier, 2006; Donaldson and Jackson, 2011; Jackson and Bouvet, 2014; Sztul et al, 2019)
Among twenty-nine members of the Arf small GTPase family in humans, including five Arf GTPases, twenty Arl GTPases, and two Sar1 GTPases (Sztul et al, 2019), in the current reconstitution studies on membrane tethering, we selected the two representative Arf isoforms, Arf1 and Arf6, both of which have been well-known to play key roles in regulating the secretory and endocytic trafficking pathways (D’SouzaSchorey and Chavrier, 2006; Sztul et al, 2019) and interact with putative membrane tethers or tethering factors such as coiledcoil tethering proteins and multisubunit tethering complexes (Prigent et al, 2003; Drin et al, 2008; Donaldson and Jackson, 2011). These two Arf-family proteins (Arf1 and Arf6), which share over 65% sequence identity (Figure 1A), and most of the other Arf isoforms are small monomeric globular proteins (2025 kDa) consisting of the amphipathic helices at the N-terminus and the following conserved Ras-superfamily GTPase domains (G-domains; 160– 170 residues; Figure 1B), and typically, they are further posttranslationally modified by a myristoyl group at the N-terminus, which functions as a lipid anchor required for their stable attachment on the membrane surface (Donaldson and Jackson, 2011; Sztul et al, 2019)
A free phosphate (Figure 1D), which were comparable to those of Rab5a-His12 used as the control of a tethering-active small GTPase (Figure 2) and the other human Rab-family proteins tested in our prior studies on reconstituted membrane tethering (Inoshita and Mima, 2017; Segawa et al, 2019)
Summary
Small GTPases of the Arf (ADP-ribosylation factor) family, belonging to the Ras superfamily, are known to be essential protein components for intracellular membrane trafficking in all eukaryotic endomembrane systems and, in general, to function through cooperation with their specific interactors, termed Arf effectors (D’Souza-Schorey and Chavrier, 2006; Donaldson and Jackson, 2011; Jackson and Bouvet, 2014; Sztul et al, 2019). Arf6-Mediated Membrane Tethering membrane-bound, coat protein complex-coated transport carriers (e.g., secretory and endocytic transport vesicles) at the donor membranes of subcellular compartments (D’SouzaSchorey and Chavrier, 2006; Sztul et al, 2019): Arf recruits the COPI (coat protein complex I) subunits for initiating vesicle formation in the retrograde Golgi-to-endoplasmic reticulum (ER) pathway (Spang et al, 1998; Bremser et al, 1999); Arf binds to the AP-2 adaptor complex in the clathrin-mediated endocytic pathway (Paleotti et al, 2005); and Sar1p assembles with Sec23/24p and Sec13/31p to form the COPII (coat protein complex II) coat and thereby promote cargo sorting and vesicle budding in the anterograde ER-Golgi trafficking pathway (Matsuoka et al, 1998; Sato and Nakano, 2004, 2005) In addition to their roles in the transport carrier formation above, it should be noted that a number of Arf-family small GTPases, including Arl (Arf-like) GTPases, have been reported to physically interact with various long coiled-coil tethering proteins and multisubunit tethering complexes as a non-coat Arf effector (Donaldson and Jackson, 2011; Sztul et al, 2019), which include golgin GMAP-210 for Arf (Drin et al, 2008), the exocyst complex for Arf (Prigent et al, 2003), Golgin-97 and Golgin-245 for Arl (Lu and Hong, 2003), the GARP complex for Arl (Rosa-Ferreira et al, 2015), and the HOPS complex for Arl (Khatter et al, 2015). Using the chemicallydefined reconstitution approach, here we report that Arf, but not Arf, can directly and physically tether two distinct lipid bilayers in the absence of any other tethering factors or Arf effectors
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