Arl1 gets into the membrane remodeling business with a flippase and ArfGEF.

Small GTP-binding proteins in the ADP ribosylation factor (Arf) family are master regulators of vesicle-mediated protein transport in the secretory and endocytic pathways (1). There are multiple members of this family in eukaryotic cells, with the lineage starting with Arf1 and its siblings (Arf2–Arf5) and extending to the more distant relatives Arf6, Sar1, and Arf-like (Arl) proteins. All these proteins cycle between GTP-bound and GDP-bound states with the assistance of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins. The best known function of GTP-bound Arfs is to recruit vesicle coat proteins from the cytosol onto the appropriate membrane for the purpose of budding a transport vesicle. However, a few links between Arfs and lipid modifying events have been reported (1). In PNAS, Tsai et al. report a unique function for Arl1, arguably the least understood member of the Arf family, in stimulating the activity of a phospholipid flippase in the trans-Golgi network (TGN) of budding yeast (2). Remarkably, this function of Arl1 also requires interaction with an ArfGEF (not an ArlGEF), infusing a bit of intrigue into the Arf family tree. These interactions are shown to be important for protein transport from the Golgi and the establishment of membrane asymmetry (2) (Fig. 1). The phospholipid flippase at the center of this regulatory circuit is Drs2, a founding member of another large protein family called type IV P-type ATPases (P4-ATPases) (3). P4-ATPases are distant relatives of cation pumps that establish ion gradients across cell membranes, but these flippases are a eukaryotic-specific lineage that evolved the ability to pump specific phospholipid molecules across the membrane bilayer rather than cations. Drs2, for example, flips phosphatidylserine (PS) and phosphatidylethanolamine unidirectionally from the luminal leaflet of the TGN to the cytosolic leaflet. This flippase activity creates a phospholipid gradient intrinsic to the membrane bilayer with PS and phosphatidylethanolamine enriched in the cytosolic leaflet, a structural organization that is maintained as membrane moves via vesicular (or tubular) transport intermediates to the plasma membrane. The phospholipid gradient in the plasma membrane can be used as a signaling platform much as ion gradients are used in signaling. Breaking of membrane asymmetry to expose PS in the outer leaflet, through activation of a “scramblase,” is critical in blood clotting cascades and is a signature feature of apoptotic cells (4). Another important consideration is that PS is a major anionic lipid in the plasma membrane. Concentration of PS in the cytosolic leaflet greatly impacts recruitment of peripheral membrane proteins to this surface and influences integral membrane protein function (5). The physiological importance of the P4ATPases is also coming into focus. There are at least 14 P4-ATPases in mammals and Drs2 orthologues, ATP8A1 and ATP8A2, are critically important for neuronal function. Mutations in ATP8A2 have recently been linked to cerebellar ataxia, mental retardation, and disequilibrium syndrome in humans and the axonal neurodegenerative and early death phenotypes of the wabbler-lethal mouse (6, 7). Loss of Atp8a1 in mice also causes neurological deficits, and the double mutant (Atp8a1 Atp8a2) is reported to die shortly after birth (7, 8). Loss of ATP8B1 function is linked to severe liver disease in humans whereas other P4-ATPases are linked to B-cell development, hearing, male fertility, obesity, and type 2 diabetes in mice. Budding yeast express five P4-ATPases, with onemember individually essential for viability (Neo1) and the other four (Drs2, Dnf1, Dnf2, Dnf3) collectively essential for viability and establishing membrane asymmetry (3). Membrane asymmetry is only half of the P4-ATPase story as these proteins, like Arfs andArls, play crucial roles in vesicle-mediated protein transport (3). It is thought that the unidirectional flip of phospholipid produces curvature in the membrane by expanding the cytosolic leaflet surface area at the expense of the luminal leaflet. This stress on the membrane would induce bending into the cytosol, making it easier for coat proteins to deform themembrane into a tightly curved vesicle (9). Arfs can also induce curvature in the membrane as their N-terminal amphipathic helices insert into the cytosolic leaflet upon GTP binding, further expanding this leaflet. Intrinsically curved proteins recruited to the membrane by Arf family members, such as coat proteins (e.g., COPI, COPII, and clathrin) and coat accessory proteins with BAR domains or amphipathic helices may also contribute to curvature and select cargo for incorporation into the forming vesicles (1). Arl proteins are known to contribute to protein trafficking events at the TGN, although the precise role of Arl1 has been elusive (1). A few effectors are known to require Arl1-GTP for recruitment to the yeast TGN, including the coiled-coil protein Imh1 (through its GRIP domain) and a clathrin adaptor (GGA) (10, 11). However, these effectors do not seem to explain the full spectrum of Arl1 function at the Fig. 1. Interaction of Arl1 with the ArfGEF Gea2 and flippase Drs2 stimulates PS translocation at the TGN. Arl1 must be in its GTP-bound conformation to bind the N-terminal cytosolic tail of Drs2 and the N-terminal region of Gea2. These interactions stabilize Gea2 interaction with the C-terminal tail of Drs2 and stimulate flippase activity. PS is flipped to the cytosolic leaflet to generate an asymmetric membrane structure. Arl1–GTP and Drs2 flippase activity is required for recruitment of Imh1 via its GRIP domain. These events likely contribute to the budding of a vesicle that transports Gas1 to the plasma membrane.