Poster for the 13th international ATPase conference, Pacific Grove, CA, USA.Poster til the 13th international ATPase conference, Pacific Grove, CA, USA
ATP8A2 is a P4-ATPase, also called a flippase, which exists as a complex with its β-subunit CDC50A. This complex preferentially transports phosphatidylserine (PS) from the exoplasmic to the cytoplasmic leaflet of biological membranes. Our recent results show that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp416) in the P-type ATPase signature sequence and exists in E1P and E2P forms, similar to Na+,K+-ATPase and Ca2+-ATPase. The mechanism of ATP8A2 resembles that of the well-characterized cation transporting P-type ATPases, as transported aminophospholipids activate the dephosphorylation directly, similar to K+ activation of dephosphorylation in Na+,K+-ATPase. By sequence alignment with well-characterized P-type ATPases, we have identified and mutated a series of strategically placed residues in the membrane domain of ATP8A2, which could be speculated to be involved in phospholipid binding. We have used the properties of mutant phosphoenzymes to examine the partial transport cycle reaction steps to elucidate the roles of these conserved residues, focusing on the fourth transmembrane segment M4. Here, Ile364 of ATP8A2 is a conserved hydrophobic flippase residue that aligns with a conserved charged glutamate residue of the classic P-type ATPases, which is involved in cation binding from both the exoplasmic and the cytoplasmic side. In contrast to other ATP8A2 examined, the ATPase activity of mutants I364A and I364S as a function of PS concentration displays an inhibition phase following the usual activation phase. This could imply that I364 is involved in releasing PS, supposedly either by pushing the phosphate headgroup or by pulling the lipid tail. Further studies of I364A, I364S and six other M4 mutants will be presented. These studies form a basis for further understanding lipid transport by this critical yet poorly understood class of P-type ATPases.