PKA/AKAP1 recruits Drp1 into large, slow turnover complexes by phosphorylating SerPKA.

(A–C) HeLa cells co-expressing wild-type or SPKAA-mutant GFP-Drp1 and either wild-type or PKA-binding deficient (ΔPKA) AKAP1 were subjected to FRAP analysis. (A) Frames showing time lapse series of representative cells (green: GFP-Drp1, red: MitoTracker Deep Red) with enlargements of the 180 s frame demonstrating recovery of Drp1 into mitochondrial foci (arrows). (B) Average recovery curves of cells expressing wild-type GFP-Drp1 and either wild-type or mutant AKAP1, and (C) plots mitochondrial Drp1 turnover as the 50% recovery time calculated from biexponential fits of individual recovery curves (R2∼0.992; means ± s.e.m. of 5–9 cells each from a representative experiment). (D) COS cells cotransfected with Drp1 and either omGFP or omPKA were treated for 5 min with the indicated concentration of the reversible, membrane permeant crosslinker dithiobismaleimidoethane (DTME), and cleared cell lysates were subjected to ultracentrifugation to sediment Drp1 complexes.