Jain, A.; von der Malsburg, A.; Götz, C.; Elmofty, M.; Reinhard, J.; Haberkant, P.; Helms, V.; Lorent, J. H.; Ernst, R.

The endoplasmic reticulum (ER) forms a dynamic network of sheets and tubules, whose molecular lipid composition remains incompletely defined. Using an optimized MemPrep workflow, we establish a high-confidence lipidome of the mammalian ER and selectively enrich membrane vesicles originating from its major structural subdomains. Quantitative lipidomics show that ER membranes are dominated by phosphatidylcholine and mono-unsaturated glycerophospholipids, consistent with a highly compressible bilayer. Although proteomics reveals a clear segregation of sheet- and tubule-associated proteins in line with a functional specialization, their lipidomes are nearly identical, indicating that these principal ER architectures share a common lipid environment. Integration of lipidomic data with bioinformatic analyses of transmembrane helices further demonstrates that the physicochemical features of ER lipids mirror those of ER-resident membrane proteins, including reduced hydrophobicity and increased polarity compared to plasma membrane proteins. These findings support a coordinated evolution of ER proteins and lipids based on shared biophysical constraints. Together, this work provides a definitive characterization of the mammalian ER lipidome and clarifies how conserved membrane properties are maintained across structurally distinct ER subdomains.