Figure 2.

An engineered mitochondria-targeted SAC1 is active in cells. (A) SAC1^mito co-localizes with the mitochondrial marker, CAX8A^N29×2-mCherry (pink). Images show representative equatorial confocal sections of two COS-7 cells. (B) Mitochondrial PI4P synthesis was induced by the addition of rapamycin, which induces dimerization of over-expressed mCherry-FKBP-PI4K^C1001 with iRFP-FRB-mito; this initiates ectopic PI4P synthesis on mitochondria outer membranes, detected with EGFP-P4Mx1 biosensor. The accumulation is blocked by co-expression of tagBFP2-SAC1^mito. Images show equatorial confocal sections of COS-7 cells before or after addition of rapamycin. P4Mx1 intensity is normalized to the average cell intensity, as quantified in B. (C) PI4P accumulation on mitochondria is blocked by SAC1^mito, despite equally efficient PI4KA^C1001 recruitment (inset). (D) The slightly decreased baseline level of PI4P at mitochondria is not statistically significant by nested t-test. Smaller green points show individual cells (7–11 cells/experiment) and larger colored-shapes show means of each of four experiments. Grand means ± SEM are also indicated. (E) Complete reduction of mitochondrial PI4P synthesis, demonstrated by a grand mean of the area under the curve for SAC1^mito-expressing cells of 0.24 ± 0.20 that is not significantly different from 0 by t-test.