Interorganelle lipid flux revealed by enzymatic mass tagging in vivo

The distinct activities of cellular organelles are dependent on the proper function of their membranes. Coordinated membrane biogenesis of the different organelles necessitates interorganelle transport of lipids from their site of synthesis to their destination membranes. Several proteins and trafficking pathways have been proposed to participate in lipid distribution, but despite the basic importance of this process, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. An obvious reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside living cells. In this strategy, two enzymes, one directed to a "donor" and the other to an "acceptor" organelle, add two distinct mass tags to lipids. Mass spectrometry-based detection of lipids bearing the two mass tags is then used as a proxy for lipid exchange between the two organelles. By applying this approach to ER and mitochondria, we show that the ERMES and Vps13-Mcp1 complexes have lipid transport activity in vivo, and unravel their relative contributions to ER-mitochondria lipid exchange.