Figure 6.

Interaction of SS-31 with isolated mitochondria. A, [ald]SS-31 binding with differing phospholipid composition. Left, percentages of total phospholipid of each species (phosphatidylcholine (PC), phosphatidylethanolamine (PE), cardiolipin (CL), monolyso-CL (MLCL), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylcholine (LPC)) in mitochondria from WT, Δtaz1, and Δcrd1 strains measured by shotgun lipidomics. Right, binding of 1 μm [ald]SS-31 with mitochondria isolated from WT, Δtaz1, and Δcrd1 yeast strains quantified as the mean fractional increase (F/F₀) in aladan emission with increasing mitochondria concentration for fully energized mitochondria. Inset, relative binding of [ald]SS-31 to mitochondria (0.2 mg ml⁻¹) in the presence or absence of 1 μm valinomycin (val) as indicated. Values shown are means ± S.D. (error bars) (n = 3). B, comparison of [ald]SS-31 binding to mitochondria and model membranes. [ald]SS-31 binding is shown as a function of CL concentration for (i) LUVs of defined TOCL composition under high-salt conditions (gray; Fig. S15) and (ii) mitochondria isolated from yeast WT strain (red; Fig. S16 and Fig. 6A). Calculation of [CL] for LUVs accounts for outer leaflet (accessible) CL only from known lipid concentrations; calculation of [CL] for mitochondrial samples was based on lipidomics analyses of Fig. 6A, accounting for total CL content. C, effects of SS-31 and salt cations on mitochondrial surface potential. Fractional change in ANS emission is shown for titration of mitoplasts from WT (left) and Δcrd1 (right) yeast with SS-31, CaCl₂, or KCl at the indicated concentrations. For WT samples, CaCl₂ titrations were also conducted following the addition of 20 μm SS-31 (CaCl₂ + SS-31). Insets, ANS emission data plotted as a function of log concentration for all three titrants. Values shown are means ± S.D. (error bars) (n = 3).