Cardiolipin Preferentially Partitions to the Inner Leaflet of Mixed Lipid Large Unilamellar Vesicles

Abstract

Cardiolipin (CL), an anionic phospholipid constituting 20% of the inner mitochondrial membrane (IMM) of eukaryotes, stabilizes electron transport chain (ETC) complexes and is a signaling agent in the early stages of apoptosis. For apoptosis, CL moves from the inner to the outer leaflet of the IMM via a poorly understood mechanism. Relative to cylindrically shaped lipids like dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG), cone-shaped CL should prefer the concave surfaces of lipid bilayers. Using the fluorophore, 1,1,2,2-tetrakis[4-(2trimethylammonioethoxy)phenyl]ethene, we have measured CL versus DOPG partitioning to the inner versus the outer leaflet of liposomes in mixed lipid systems with DOPC. DOPG shows no leaflet preference. However, CL has a 4:1 preference for the concave surface of the inner leaflet of liposomes. To further test the inner leaflet preference of CL, we show that cytochrome c binding to the outer convex surface of 20% CL/80% DOPC vesicles is strongly attenuated. Because the outer leaflet of intracristal regions of the IMM has a concave curvature, the preference of CL for concave surfaces may facilitate the transport of CL from the inner to the outer leaflet of the IMM needed for CL remodeling, the optimal functioning of the ETC, and signaling in the early stages of apoptosis.

Mitochondrial Cristae Architecture and Functions: Lessons from Minimal Model Systems

“…In line with this last assumption and the conclusions of Beltran-Heredia’s paper [ 80 ], a recent paper [ 125 ] studied CL distribution between the bilayer leaflets of large unilamellar liposomes (LUV, 100 nm diameter). They use the fluorophore TTAPE-Me (1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)-phenyl]ethene), which fluoresces upon binding to anionic head groups of lipids, but especially CL.…”

“…The LUV have a diameter of 100 nm and are made of CL/PC with different CL concentrations. Reproduced with permission from Elmer-Dixon et al [ 125 ], J. Phys. Chem.…”

TTAPE-Me dye is not selective to cardiolipin and responds to main anionic phospholipids unspecifically

“…Figure 2A and B show that the shapes of emission spectra for pure CL, PG and PS as well as for their binary mixtures with PE are indistinguishable. It is necessary to note that some published experiments [28] were performed in the presence of drastic excess of the probe and the background signal was not subtracted properly from the emission spectra. In such cases spurious differences in the shapes of spectra could be interpreted incorrectly as apparent selectivity of TTAPE-Me to particular lipids.…”

“…Recently, a water soluble TTAPE-Me dye was proposed as a promising CL-specific fluorescent probe suitable for CL detection and sidedness studies [27,28]. TTAPE-Me is non-fluorescent in aqueous solutions.…”

“…It was reported recently that TTAPE-Me responds similarly to PG and CL [28], but these experiments were performed in the presence of drastic excess of the probe. As a result it is not known if TTAPE-Me is suitable for CL detection in PG-rich bacterial membranes and mammalian plasma membranes where PS is one of the most abundant lipids.…”