From the Cover: PNAS Plus: Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p

ADP ribosylation factors (Arfs) are the central regulators of vesicle trafficking from the Golgi complex. Activated Arfs facilitate vesicle formation through stimulating coat assembly, activating lipid-modifying enzymes and recruiting tethers and other effectors. Lipid translocases (flippases) have been implicated in vesicle formation through the generation of membrane curvature. Although there is no evidence that Arfs directly regulate flippase activity, an Arf-guanine-nucleotide-exchange factor (GEF) Gea2p has been shown to bind to and stimulate the activity of the flippase Drs2p. Here, we provide evidence for the interaction and activation of Drs2p by Arf-like protein Arl1p in yeast. We observed that Arl1p, Drs2p and Gea2p form a complex through direct interaction with each other, and each interaction is necessary for the stability of the complex and is indispensable for flippase activity. Furthermore, we show that this Arl1p-Drs2p-Gea2p complex is specifically required for recruiting golgin Imh1p to the Golgi. Our results demonstrate that activated Arl1p can promote the spatial modulation of membrane organization at the trans-Golgi network through interacting with the effectors Gea2p and Drs2p.ADP ribosylation factors (Arf)/SARs, a subfamily of the Ras small GTP-binding protein superfamily, are critical regulators of vesicular trafficking in eukaryotic cells (1–3). Like Ras, Arf and Arf-like (Arl) proteins cycle between the inactive GDP-bound form and active GTP-bound forms to carry out their functions. The conversion from the GDP-bound to the GTP-bound form is facilitated by Arf guanine-nucleotide-exchange factor (Arf-GEF), whereas GTP hydrolysis is catalyzed by an Arf GTPase-activating protein (Arf-GAP) (4, 5). Activated Arf recruits numerous proteins to the membrane or activates lipid-modifying enzymes to facilitate vesicle formation, including vesicle coat assembly, membrane curvature generation, lipid composition alteration, and final membrane fission (4, 6–8).Arl1p is the best-studied Arl protein and is expressed in organisms ranging from yeast to humans (9, 10). Both yeast Arl1p and human Arl1 localize to the trans-Golgi network (TGN), where they regulate multiple membrane-trafficking pathways (9, 11, 12). Yeast Arl1p participates in three different pathways at the TGN, including glycosylphosphatidylinositol (GPI)-anchored protein transport, clathrin adaptor protein Gga recruitment, and targeting of the GRIP domain–containing protein Imh1p to the Golgi. In addition to Imh1p, the Arf-GEF–like protein, Mon2p (also called Ysl2p), and the clathrin adaptor protein, Gga2p, directly interact with Arl1p and are considered to be Arl1p effectors (13).Arf-GEFs catalyze the GTP binding of Arf/Arl through the conserved Sec7 domain and are upstream regulators of Arf/Arl activities (14, 15). In yeast, the human GBF1 homolog Gea2p, which functions redundantly with Gea1p, displays Arf-GEF activity toward Arf1p and is believed to be involved in ER-Golgi and intra-Golgi transport (16, 17). Recently, Gea2p was shown to directly interact with the TGN-localized aminophospholipid translocase Drs2p and to stimulate its flippase activity (18, 19). Similarly, it was reported that the Arf-GEF–like protein Mon2p interacted with both Arl1p and the Drs2p family protein Neo1p and that the Mon2-Neo1-Arl1 complex cooperated to regulate the Golgi recruitment of Gga2p (13). These results suggest that Arf-GEFs may act with members of the Drs2p subfamily of P-type ATPases to facilitate lipid translocation. However, the role of Gea2p in Drs2p-mediated flippase activity is not clear.Flippases translocate specific phospholipid molecules to establish the asymmetry of membranes (19–22). Therefore, flippases can drive membrane bending toward the cytosol and contribute to vesicle-mediated protein transport by facilitating the generation of membrane curvature that is inherent to this process (23). The inactivation of Drs2p results in defects in the formation of an Arf/clathrin-dependent class of post-Golgi vesicles that carry exocytic cargo (24). Moreover, a mutation in DRS2 was shown to cause synthetic lethality with clathrin heavy chain and ARF1 (25). These observations indicate the importance of flippase activity for vesicle trafficking, and Drs2p is involved in protein transport at the TGN.To explore the possibility that Arl1p might, like Arf proteins, play a role in regulating membrane dynamics, we use an analysis of both physical interaction and functional interaction to demonstrate that Drs2p and Gea2p are effectors of Arl1p. We find that these proteins—Arl1p, Drs2p, and Gea2p—are all required to form a stable complex to stimulate Drs2p flippase activity and complete certain functions of Arl1p. Our study provides insight into the molecular mechanisms of spatial membrane dynamics at the TGN and their regulation by the Arl1p-Gea2p-Drs2p cooperating network.