In vivo analyses reveal rapid and permissive lipid transport between the ER and mitochondria

Interorganelle lipid transport is essential for mitochondrial membrane biogenesis and function, yet its kinetics and substrate selectivity remain poorly understood in vivo . Here, we developed two complementary approaches to quantify lipid trafficking from the endoplasmic reticulum (ER) to mitochondria in yeast. Metabolic labeling combined with organelle fractionation revealed that newly synthesized phospholipids rapidly accumulate in mitochondria, with 20-35% of newly synthesized molecules detected in mitochondrial fractions within minutes of synthesis. To directly quantify lipid flux, we established a synthetic transport assay based on the production of heterologous galactolipids absent from yeast. This approach revealed an ER-to-mitochondria transport flux of approximately 2.6 x 10 ⁵ lipid molecules per cell per minute. Remarkably, galactolipids were transported with high efficiency despite their absence from fungal membranes, indicating limited substrate selectivity of ER-mitochondria lipid transport pathways. Together, these complementary assays provide quantitative tools to investigate intracellular lipid transport and reveal the rapid and permissive nature of lipid exchange between the ER and mitochondria.