Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P 4 -ATPase ATP8A2
- 17 January 2012
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 109 (5), 1449-1454
- https://doi.org/10.1073/pnas.1108862109
Abstract
ATP8A2 is a P(4)-ATPase ("flippase") located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P(4)-ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp(416)) in the P-type ATPase signature sequence and exists in E(1)P and E(2)P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp(196), Glu(198), Lys(873), and Asn(874) in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E(2)P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K(+) activation of dephosphorylation in Na(+),K(+)-ATPase. Lys(873) mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys(873), located in transmembrane segment M5 at a "hot spot" for cation binding in Ca(2+)-ATPase and Na(+),K(+)-ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys(865) is unimportant for aminophospholipid sensitivity. Binding of Na(+), H(+), K(+), Cl(-), or Ca(2+) to the E(1) form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.Keywords
This publication has 28 references indexed in Scilit:
- Critical Role of the β-Subunit CDC50A in the Stable Expression, Assembly, Subcellular Localization, and Lipid Transport Activity of the P4-ATPase ATP8A2Journal of Biological Chemistry, 2011
- Heteromeric Interactions Required for Abundance and Subcellular Localization of Human CDC50 Proteins and Class 1 P4-ATPasesJournal of Biological Chemistry, 2010
- Disruption of the ATP8A2 gene in a patient with a t(10;13) de novo balanced translocation and a severe neurological phenotypeEuropean Journal of Human Genetics, 2010
- The Rapid-onset Dystonia Parkinsonism Mutation D923N of the Na+,K+-ATPase α3 Isoform Disrupts Na+ Interaction at the Third Na+ SitePublished by Elsevier BV ,2010
- Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc MembranesJournal of Biological Chemistry, 2009
- Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeastProceedings of the National Academy of Sciences, 2009
- ATP8B1 is essential for maintaining normal hearingProceedings of the National Academy of Sciences, 2009
- How Ca2+-ATPase pumps ions across the sarcoplasmic reticulum membraneBiochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008
- Mutagenesis of Segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of Sarcoplasmic Reticulum Ca2+-ATPase Produces Pumps Defective in ATP BindingPublished by Elsevier BV ,1996
- A Glu329→ Gln variant of the α-subunit of the rat kidney Na+K+-ATPase can sustain active transport of Na+and K+and Na+K+-activated ATP hydrolysis with normal turnover numberFEBS Letters, 1993