Following anterograde transport of phosphatidylserine in yeast in real time

Abstract

In order to understand how lipids are sorted between cellular compartments, kinetic assays are required to selectively follow the transport of lipid species in cells. We present here a microfluidics-based protocol to follow the transport of phosphatidylserine (PS) in yeast cells from the site of its synthesis, the endoplasmic reticulum (ER), to downstream compartments, primarily the plasma membrane under our conditions. This assay takes advantage of yeast cells lacking Cho1, the enzyme responsible for PS synthesis. Lyso-PS can be added exogenously and is taken up by the cells and converted to PS. Because acylation of lyso-PS to PS appears to occur at the ER, anterograde transport of PS from the ER can then be followed by fluorescent microscopy using the specific PS reporter C2Lact-GFP. We describe the construction of the required cho1Δ yeast strain and the preparation of lyso-PS. We present an example of the use of this assay to follow the activity of the yeast PS transport proteins Osh6 and Osh7.