MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress. Synopsis: This study describes a new technology for isolating organelle membranes from yeast, MemPrep. Combined with quantitative lipidomics, MemPrep establishes molecular fingerprints of membrane-based forms of endoplasmic reticulum (ER) stress. MemPrep provides quantitative insight into the compositions of the yeast vacuole and ER membrane. The ER membrane composition is significantly distinct from the whole cell lipidome. Lipid metabolic perturbation causes dramatic changes of the ER membrane composition, which sometimes trigger ER stress. Inositol depletion and prolonged proteotoxic stress lead to distinct lipid fingerprints and membrane proteomes. Increased membrane thickness and low levels of anionic lipids are common denominators of lipid bilayer stress. Combined with proteomics and quantitative lipidomics, this method for purification of specific yeast membranes establishes molecular fingerprints of membrane-based forms of endoplasmic reticulum (ER) stress. [ABSTRACT FROM AUTHOR]