Shimanaka, Y.; Tol, M. J.; Rocha-Roa, C.; Jellinek, M. J.; Cui, L.; Bender, A.; Bedard, A. H.; Milner, M. G.; Melillo, B.; Shoucri, B. M.; Wong, A.; Williams, K. J.; Stiles, L.; Shum, M.; Weston, T. A.; Cohn, W.; Whitelegge, J. P.; Budin, I.; Bertholet, A. M.; Cravatt, B. F.; Young, S. G.; Houten, S. M.; Argmann, C.; Ford, D. A.; Liesa, M.; Shirihai, O. S.; Vanni, S.; Tontonoz, P.

Cold stress elicits dynamic remodeling of the mitochondrial lipidome in brown adipose tissue (BAT), marked by an increase in arachidonoyl-phosphatidylethanolamine (AA-PE). However, the function of membrane lipid rewiring in thermoregulatory physiology has been a longstanding mystery. Here, we identify LPCAT3 as a cold-regulated O-acyltransferase driving the highly selective accrual of AA-PE in BAT mitochondria. Lipid-based proteomics, molecular dynamics simulations, and bioenergetic analyses reveal that AA-PE partitions at the COX4I1 interface of the Cytochrome c oxidase complex, enhancing electron transport chain (ETC) efficiency. Accordingly, fat-specific Lpcat3-knockout mice have defects in respiratory-dependent BAT thermogenesis and cold tolerance, despite intact {beta}-adrenergic signaling and UCP1 function. Under cold acclimation, Lpcat3-/- BAT exhibits ETC dysfunction and activation of the integrated stress-response. Thus, our study illuminates a cold-regulated lipid--protein interaction as a gating factor in UCP1-dependent thermogenesis.