The
P-type ATPase protein family includes, in addition to
ion pumps such as Ca2+-
ATPase and Na+,K+-
ATPase, also
phospholipid flippases that transfer
phospholipids between membrane leaflets.
P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the
protein. The large size of the
lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the
lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the
lipid substrate to only a few M5-M6 residues. The M6
asparagine Asn-905 stood out as being essential for the
lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the
lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of
P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the
neurological disorder cerebellar ataxia,
mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 (i.e. V906A/E/F/L/Q/S) suggest that the
lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the
ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase
protein.