Cell-Based Lipid Flippase Assay Employing Fluorescent Lipid Derivatives

P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, stud...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1377; p. 371
Main Authors Jensen, Maria S, Costa, Sara, Günther-Pomorski, Thomas, López-Marqués, Rosa L
Format Journal Article
LanguageEnglish
Published United States 2016
Subjects
Azoles - chemistry
Enzyme Assays - methods
Flow Cytometry
Fluorescent Dyes - metabolism
Lipid Metabolism
Lipids - chemistry
Nitrobenzenes - chemistry
Phospholipid Transfer Proteins - metabolism
Saccharomyces cerevisiae - cytology
Phospholipid probes
Fluorescence
Flow cytometry
Back-exchange
Saccharomyces cerevisiae
Heterologous expression
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Summary:P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid flippase activities in the plasma membrane of cells, using yeast as an example.
ISSN:1940-6029
DOI:10.1007/978-1-4939-3179-8_33