PA alone is sufficient for PILS-Nir1 membrane binding. (A) FKBP-PI4K and FKBP-PIP5K were co-expressed in HEK293A cells to convert PI into PI4P and then PIP₂ after the dimerization of the FKBP fragment and mitochondrial-localized FRB with 1 µM rapamycin. Cells expressing FKBP-PI4K alone were used to determine any effects of PI4P on PILS-Nir1 and NES-PABDx2-Spo20 membrane binding. (B) Quantification and representative images of FKBP recruitment and PIP₂ production as monitored by the PIP₂ biosensor PH-PLC δ1. (C) Localization of PILS-Nir1 and NES-PABDx2-Spo20 was unchanged upon ectopic PI4P and PIP₂ production. (D) FKBP-PI-PLC and FKBP-DGKα were co-expressed in cells and recruited to mitochondria to produce DAG from PI and subsequently produce PA from DAG. Control cells expressed FKBP-PI-PLC alone to look at the effects of DAG production on the PA biosensors. (E) Quantification and representative images of FKBP recruitment. (F) PILS-Nir1 and NES-PABDx2-Spo20 were only recruited to mitochondria where PA was produced by FKBP-DGKα. All experiments were performed three to four independent times, with the xy graphs showing the grand means of the experiments ± SEM. 33–45 total cells were analyzed. Note the PILS-Nir1–expressing cell shown in F is the same as shown in E.