Application of image cytometry to characterize heterologous lipid flippases in yeast.
Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number of lipid flippases has been identified but key features of their activity remain to be elucidated. A well-established method to characterize ATP-driven flippases is based on their heterologous expression in yeast, followed by incubation of the cells with fluorescent lipids. Internalization of these probes is typically monitored by flow cytometry, a costly and maintenance-intensive method. Here, we have optimized a protocol to use an automated image-based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical flow cytometric evaluation of lipid-labeled cells. In addition, we demonstrated that expression of fluorescently tagged flippase complexes can be directly co-related with fluorescent lipid uptake using the image-based cell counter system. The method extends the number of techniques available for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry.
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Maria S
MS
Jensen
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Sara R
SR
Costa
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Lisa
L
Theorin
Institute for Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.
Jan Pravsgaard
JP
Christensen
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.|Faculty of Chemistry and Biochemistry, Department of Molecular Biochemistry, Ruhr University Bochum, Universitätstrasse 150, Bochum, D-44780, Germany.
Thomas Günther
TG
Pomorski
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Rosa L
RL
López-Marqués
D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D005434:Flow Cytometry; D019044:Image Cytometry / Q000379:methods*; D008055:Lipids; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D008055:Lipids; D029701:Saccharomyces cerevisiae Proteins
Key terms: yeast; NBD-lipid uptake; automated cell counter; cytometry; flippases; flow cytometer