Abstract
Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana, in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2–ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise, the enzymatic activity of the ALA2–ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase.
Highlights
PPurpose oli 2078 oli 2953 oli 2954 oli 2390 oli3422 oli3423
GTAGAGACTGCAGCTTTAGATGGTCAAACTGATCTCAA AACAAGAGTG CACTCTTGTTTTGAGATCAGTTTGACCATCTAAAGCTGC AGTCTCTAC ATTGCATGCATGTACCCATACGATGTTCCAGATTACGCT GAATTTTCTTCCGAAGAACTTGTGG GAATTCTTATGAAGCGTTTTGTGTACATTAACG
Fwd ALA2 start (SphI, HA-tag) Fwd on ALA2 start (BamHI) Rvs ALA2 stop (SacI)
Summary
PPurpose oli 2078 oli 2953 oli 2954 oli 2390 oli3422 oli3423 GTAGAGACTGCAGCTTTAGATGGTCAAACTGATCTCAA AACAAGAGTG CACTCTTGTTTTGAGATCAGTTTGACCATCTAAAGCTGC AGTCTCTAC ATTGCATGCATGTACCCATACGATGTTCCAGATTACGCT GAATTTTCTTCCGAAGAACTTGTGG GAATTCTTATGAAGCGTTTTGTGTACATTAACG
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