Coleman, Jonathan Allan2; Vestergaard, Anna Lindeløv4; Molday, Robert S.2; Vilsen, Bente4; Andersen, Jens Peter4
1 Department of Biomedicine - Physiology and Biophysics, Department of Biomedicine, Health, Aarhus University2 University of British Columbia, Vancouver3 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University4 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University
Poster for the 13th international ATPase conference, Pacific Grove, CA, USA
ATP8A2 is a P4-ATPase ("lipid flippase") highly expressed in the retina, brain, and testes. Previous studies in our laboratory have shown that ATP8A2 exists as a complex with its β-subunit CDC50A in retinal rod and cone photoreceptor cells and this complex preferentially transports phosphatidylserine (PS) from the lumen to the cytoplasmic leaflet of photoreceptor outer segment disk membranes. A crucial question is whether the mechanism of lipid transport by P4-ATPases is similar to the well characterized ion transporting P-type ATPases. Detailed kinetic studies have demonstrated that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate residue of the DKTG motif. We have found that the phosphoenzyme exists in two distinct forms, E1P and E2P, similar to other P-type ATPases and have used this property to understand the partial reactions of the transport cycle. We have examined the function of conserved residues Asp196, Glu198, and Lys873. Asp196 and Glu198 located in the T/D-G-E-T/S motif of the A-domain are important for dephosphorylation of E2P with the glutamate being essential. PS activates dephosphorylation of ATP8A2 in a manner analogous to the dephosphorylation of the Na+/K+-ATPase mediated by K+. Lys873 located in the membrane domain is a key residue involved in lipid binding and transport. Counterion binding to the E1 form has not be observed under various ionic conditions, suggesting that ion binding is not required for phosphorylation and that phospholipids are likely the only species which are transported by P4-ATPases. These studies form a basis for further understanding lipid transport by this critical yet poorly understood class of P-type ATPases.
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13th international ATPase conference Na,K-ATPase and related ATPases, 2011