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

Roland BP, Graham TR
Proc Natl Acad Sci U S A. 2016 113 (31): E4460-6

PMID: 27432949 · PMCID: PMC4978280 · DOI:10.1073/pnas.1525730113

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 glycerophospholipids 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.

MeSH Terms (14)

Adenosine Triphosphatases Amino Acid Sequence Asparagine ATP-Binding Cassette Transporters Biological Transport Cell Membrane Directed Molecular Evolution Gain of Function Mutation Mutagenesis Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Sequence Homology, Amino Acid Sphingomyelins Substrate Specificity

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