Analysis of Amino Acid Residues of Potential Importance for Phosphati-dylserine Specificity of P4-type ATPase ATP8A2

Research output: Contribution to conferencePosterResearch

  • Louise Mogensen, Denmark
  • Anna Lindeløv Vestergaard, Denmark
  • Stine Mikkelsen
  • Jonathan Allan Coleman, Denmark
  • Bente Vilsen
  • Robert S. Molday, Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, BC, Canada
  • Jens Peter Andersen
The asymmetric structure of the plasma membrane is maintained through internalization of phos-pholipids by the family of P4-ATPases by a poorly characterized mechanism. Studies in yeast point towards a non-classical pathway involving important residues of a two-gate mechanism [1]. Glycine-230 and alanine-231 of Dnf1 have shown to be important determinants of the phosphatidylcholine headgroup specificity of a putative entry gate, as seen by substitution of these residues by two glutamines as present in the phosphatidylserine-specific Drs2 at the same position. Aspargine-550 also appeared to contribute to the phospholipid specificity of Dnf1 and was proposed to participate in an exit gate[1].
Through site-directed mutagenesis studies, we have examined the possibility of a two-gate mechanism in the phosphatidylserine-specific mammalian flippase ATP8A2. Its homologous resi-dues glutamine 95 and 96 has been replaced by glycine (Q95G) and alanine (Q96A), singly or to-gether in a double mutant (Q95GQ96A), to evaluate their roles in connection with substrate entry at the exoplasmic side of ATP8A2. The putative exit gate at the cytoplasmic side was examined by replacing the asparagine at position 302 of ATP8A2 with alanine (N302A), tyrosine (N302Y) and serine (N302S). Furthermore, a triple mutant of ATP8A2 (Q95GQ96AN302S) was studied to reveal any cooperativity between the two gates, as observed in yeast [1]. The affinities of the mutants for phosphatidylserine and phosphatidylethanolamine together with their maximal velocity have been analyzed by an ATPase activity assay and compared with those of the wild type ATP8A2 protein. Also, partial reaction steps of the catalytic cycle of these ATP8A2 mutants were studied by phos-phorylation assays. Our results indicate both similarities and differences between ATP8A2 and the yeast flippases. The results supplement recent studies of the ATP8A2 flippase revealing a hydro-phobic gate that facilitates the transport along a water-filled pathway in the protein transmembrane domain[2].

1. Baldridge, R.D. and T.R. Graham, Proceedings of the National Academy of Sciences, 2013. 110(5): p. E358-E367.
2. Vestergaard, A.L., J.A. Coleman, T. Lemmin, S.A. Mikkelsen, L.L. Molday, B. Vilsen, R.S. Molday, M. Dal Peraro, and J.P. Andersen, Proc Natl Acad Sci U S A, 2014. 111(14): p. E1334-43.

Original languageEnglish
Publication yearAug 2014
Number of pages1
Publication statusPublished - Aug 2014
Event14th International ATPase Conference “Na,K‐ATPase and related transport ATPases: Structure, mechanism, cell biology, health and disease” - Conference Center De Werelt, Lunteren, Netherlands
Duration: 30 Aug 20145 Sep 2014


Conference14th International ATPase Conference “Na,K‐ATPase and related transport ATPases: Structure, mechanism, cell biology, health and disease”
LocationConference Center De Werelt

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