N905 of TM6 is crucial to the function of the mammalian flippase ATP8A2: Poster at the 15th International Conference on Na,K-ATPase and Related Transport ATPases, Otsu, Japan, 2017

Publikation: KonferencebidragPosterForskning

P-type ATPases comprise in addition to ion pumps a branch denoted P4-type ATPases or “flippases”, which transport large phospholipids instead of inorganic ions, thereby posing the enigma of “the giant substrate problem”. In the ion pumps, transmembrane segments M5 and M6 contain residues that are directly involved in coordination of the transported ions and are central mechanistic elements. During ATP hydrolysis, movements of the cytoplasmic domains are transduced to the membrane domain making M5 and M6 highly dynamic participants in the transport of the ions through the membrane. ATP8A2 is a flippase that translocates the aminophospholipids phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic side of the plasma membrane [1]. We have here investigated the importance of M5 and M6 in ATP8A2 by aligning the residues in ATP8A2 with several of the ion-pumps to locate M5 and M6 of ATP8A2 and have replaced replacing all residues of M5 and M6 individually with alanine (alanine scanning). Each mutant was characterized functionally with respect to maximum velocity and lipid substrate affinity. Our analysis brings out a map of M5 and M6, where only a few residues are highly sensitive to mutagenesis. These were further examined by replacement with several other amino acids and by study of the individual steps of the reaction cycle of the mutants. One residue that stands out as particularly interesting is N905, which appeared to be crucial to the substrate activated dephosphorylation. N905 aligns with the ion binding residues N796 in Ca2+-ATPase and D804 in Na+,K+-ATPase. Our findings provide new evidence of mechanistic similarities between flippases and P-type ion pumps and raise the question whether the central region of the transmembrane sector encompassing M6 could be directly involved in the lipid translocation, unlike the previous suggestions of a peripheral pathway [2, 3].

REFERENCE LIST

1. Coleman, J.A., et al., J.Biol.Chem., 2009. 284(47): p. 32670-32679.
2. Baldridge, R.D., et al., Proc Natl Acad Sci U S A, 2013. 110(5): p. E358-67.
3. Vestergaard, A.L., et al., Proc.Natl.Acad.Sci.U.S.A, 2014. 111(14): p. E1334-E1343.

OriginalsprogEngelsk
Udgivelsesår24 sep. 2017
Antal sider1
StatusUdgivet - 24 sep. 2017

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