LC-MSMS with isobaric tagging on Flag-coimmunoprecipitation eluates from Flag-Pgrmc1 and Y113F Flag-Pgrmc1 expressing Pgrmc1 KO mouse liver membranes

Progesterone receptor membrane component 1 (PGRMC1) is a heme binding protein implicated in a wide range of cellular functions. Our previous studies showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and promotes activity of these enzymes. Recently, the paralog PGRMC2 was shown to function as an intracellular heme chaperone. Here, we examined the function of the Pgrmc1 by identifying binding partners for wild-type Pgrmc1 and the Pgrmc1 Y113F mutant that is defective for heme iron coordination. Pgrmc1 knockout mice were infected with AAV8 expressing either GFP, Flag-Pgrmc1, or Y113F Flag-Pgrmc1, and Flag-Pgrmc1 was affinity purified using anti-Flag antibodies from extracts of liver membranes. These experiments (1) demonstrated that Pgrmc1 binds to different cytochrome P450 enzymes and (2) revealed that Pgrmc1 Y113F specifically fails to bind to ferrochelatase.