Phospholipid flippases in membrane remodeling and transport carrier biogenesis

Abstract

Molecular mechanisms underlying the formation of multiple classes of transport carriers or vesicles from Golgi and endosomal membranes remain poorly understood. However, one theme that has emerged over three decades is the dramatic influence of membrane lipid remodeling on transport mechanisms. A large cohort of lipid transfer proteins, lipid transporters, and lipid modifying enzymes are linked to protein sorting, carrier formation and SNARE-mediated fusion events. Here, we focus on one type of lipid transporter, phospholipid flippases in the type IV P-type ATPase (P4-ATPase) family, and discuss recent advances in defining P4-ATPase influences on membrane remodeling and vesicular transport.

Figure 1.. P4-ATPase-mediated lipid transport imparts stress on membranes that can be relieved through membrane bending, induced flop of other lipids, exchange of lipid by transfer proteins, or activation of a scramblase.

P4-ATPases convert the chemical potential energy in ATP to potential energy stored in a lipid gradient within the membrane. In the example shown, the flippase-catalyzed unidirectional transport of PS creates molecular crowding stress in the cytosolic leaflet, packing defect stress in the luminal leaflet, and a charge difference between the leaflets. The potential energy in the membrane can be used by the cell to drive at least four different processes. 1) Exchange of cytosolic leaflet lipids with a bulky headgroup (such as phosphatidylinositol-4-phosphate or PS) for sterol or ceramide by lipid transfer proteins can relieve molecular crowding stress and facilitate membrane remodeling in the Golgi (sphingolipid synthesis and sterol loading). 2) Membrane bending to support budding or scission of protein transport carriers. 3) Displacement of lipids capable of spontaneous flip-flop to the luminal leaflet. Cholesterol is an example of a lipid that flip-flops rapidly between leaflets in an energy-independent manner. Coupling of PS flip to the cytosolic leaflet with cholesterol flop to the luminal leaflet could play an important role in lateral segregation of sterol, sphingolipid and protein in raft-like structures for sorting into exocytic carriers. 4) Rapid exposure of PS in the outer leaflet of cells through activation of a scramblase. This signaling event plays a critical role in apoptosis, blood clotting, immune system suppression, fusion of myoblasts and bone formation.

Figure 2.. Roles for ATP9A and ATP8A1 in endosomal recycling pathways.

ATP8A1 (TAT-1 in C. elegans) and EHD1 (RME-1 in C. elegans) are required for transport of endocytosed proteins from recycling endosomes to the Golgi and for return back to the plasma membrane. Transport of PS to the cytosolic leaflet by ATP8A1 helps recruit EHD1 to recycling endosomes (RE) to potentially drive scission of tubular transport carriers. ATP9A (TAT-5 in C. elegans and Neo1 in S. cerevisiae) and Snx3-retromer is required for protein transport from early endosomes (EE) to the Golgi. MON2 and DOPEY2 are also linked to the function of ATP9A in this pathway.

Figure 3.. Model for how a PS flippase (Drs2-Cdc50) contributes to the polyubiquitin- and COPI-dependent recycling of an exocytic SNARE.

Snc1 is a t-SNARE that functions in the fusion of exocytic carriers with the plasma membrane. After endocytosis, Snc1 recycles through the endosomal system back to the Golgi for re-packaging into exocytic carriers. This recycling process requires Drs2-Cdc50, Rcy1, the ArfGAP Gcs1, COPI, and modification of Snc1 by K63-linked polyubiquitin. Rcy1 binds to a C-terminal regulatory domain on Drs2 and likely stimulates flippase activity. PS flip to the cytosolic leaflet increases membrane curvature and negative charge to recruit Gcs1, which binds to both COPI and Snc1. In addition, the twin β propellers of α and β’ COP bind directly to K63-linked polyubiquitin and this interaction is essential for Snc1 recycling.