Auto-Inhibitory Mechanism for the Regulation of a P4-ATPase
Sebastian, Tessy Tereas
Vesicular transport of proteins is a process essential for cell health and viability. Integral membrane proteins, called phospholipid flippases, play important roles in the formation of transport vesicles at sites of membrane budding. Flippases also establish and maintain membrane asymmetry. This is achieved by using the energy of ATP hydrolysis to unidirectionally translocate specific phospholipid molecules from the lumenal to the cytosolic side of the membrane bilayer. It is hypothesized that the resulting imbalance in phospholipid number between the bilayers causes membrane bending, captured by coat and accessory proteins. This leads to the formation of mature cargo-containing transport vesicles. Drs2p, the founding member of the flippase (P4-ATPase) family, has been implicated in the formation of clathrin-coated vesicles at the trans-Golgi network. The influence of Drs2p flippase activity on membrane curvature is explored in this thesis. When late Golgi membranes were purified, and Drs2p on these membranes were activated by ATP addition, tubulation, elongation, and membrane fusion phenotypes were observed. Furthermore, when purified Drs2p was reconstituted into giant unilamellar vesicles (GUVs), and activated by ATP addition, preliminary data indicates the formation of spindle-like tubes. Due to the presence of purified Drs2p (protein found most abundantly in this system) and dependence of this reaction to ATP addition, it is likely this membrane tubulation is Drs2p-dependent. Thus, these data implicate Drs2p in membrane curvature formation due to the ability to translocate phospholipids across the membrane bilayer. Work is also presented that looks at the role of the C-terminal tail of Drs2p on regulation of flippase activity. The C-terminal tail auto-regulates Drs2p function and loss of auto-inhibition by the tail – either by proteolysis or interaction with Drs2p effectors – stimulates the flippase activity of Drs2p. Furthermore, phosphatidylinositol 4-phosphate is shown to have a direct influence in binding the C-tail to stimulate Drs2p activity. This is the first evidence, in a P4-ATPase, that the C-tail functions as a domain responsible for regulating flippase activity, and this regulation can be relieved by interaction with a phosphoinositide.