Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase

作者：Roland Bartholomew P; Graham Todd R

来源：Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(31): E4460-E4466.

DOI：10.1073/pnas.1525730113

• sphingomyelin
• P4-ATPase
• membrane asymmetry
• directed evolution

Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophos-pholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.

• 出版日期2016-8-2