Fig. 3. CL-dependent Aac2 oligomerization cannot be rescued with protein-stabilizing Aac2 inhibitors.

(A) Yeast strains were grown in YP-Sucrose medium supplemented with ³²P (2.5 μCi/ml) overnight. Phospholipids were extracted and separated by thin-layer chromatography (TLC). The migration of PC, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidic acid (PA), and CL is indicated (n = 6). (B) WT or crd1∆ mitochondria (250 μg), preincubated with CATR (40 μM) or BKA (10 μM) as listed, were solubilized with 1.5% (w/v) digitonin and FLAG-Aac2 immunoprecipitated using anti-FLAG resin. The presence of copurified HA-Aac2 and subunits of complexes III (Cor1, Cor2, Rip1, and Qcr6) and IV (Cox1 and Cox4) was determined by immunoblotting; Atp1, Atp2, and Kgd1 served as controls. *, nonspecific bands. Proteins that copurified with FLAG-Aac2 when CL-containing mitochondria were preincubated with CATR (red arrowheads) or BKA (blue arrowheads) are marked. Four percent of input (mitochondria) and unbound (flow through following FLAG immunoprecipitation) was analyzed (n = 4). (C) The amount of HA-Aac2 and respiratory complex subunits coimmunoprecipitated with FLAG-Aac2 in BKA or CATR pretreated CL-null mitochondria was determined relative to similarly treated CL-containing mitochondria (means ± SEM for n = 4 independent experiments). Statistical differences were determined by Mann-Whitney rank sum test.