Cardiolipin-dependent reconstitution of respiratory supercomplexes from purified Saccharomyces cerevisiae complexes III and IV

Abstract

Here, we report for the first time in vitro reconstitution of the respiratory supercomplexes from individual complexes III and IV. Complexes III and IV were purified from Saccharomyces cerevisiae mitochondria. Complex III contained eight molecules of cardiolipin, and complex IV contained two molecules of cardiolipin, as determined by electrospray ionization-mass spectrometry. Complex IV also contained Rcf1p. No supercomplexes were formed upon mixing of the purified complexes, and low amounts of the supercomplex trimer III(2)IV(1) were formed after reconstitution into proteoliposomes containing only phosphatidylcholine and phosphatidylethanolamine. Further addition of cardiolipin to the proteoliposome reconstitution mixture resulted in distinct formation of both the III(2)IV(1) supercomplex trimer and III(2)IV(2) supercomplex tetramer. No other anionic phospholipid was as effective as cardiolipin in supporting tetramer formation. Phospholipase treatment of complex IV prevented trimer formation in the absence of cardiolipin. Both trimer and tetramer formations were restored by cardiolipin. Analysis of the reconstituted tetramer by single particle electron microscopy confirmed native organization of individual complexes within the supercomplex. In conclusion, although some trimer formation occurred dependent only on tightly bound cardiolipin, tetramer formation required additional cardiolipin. This is consistent with the high cardiolipin content in the native tetramer. The dependence on cardiolipin for supercomplex formation suggests that changes in cardiolipin levels resulting from changes in physiological conditions may control the equilibrium between individual respiratory complexes and supercomplexes in vivo.

FIGURE 1.

CL species and content in the purified CIII and CIV determined by ESI-MS. Total CL was determined as the sum of the five prevalent CL species (1344 (16:1)₄; 1372 (16:1)₃(18:1)₁; 1400 (18:1)₂(16:1)₂; 1428 (18:1)₃(16:1)₁, and 1456 (18:1)₄) from values of singly ionized CL. Data represent the average of four independent runs, and the bars indicate standard deviations.

FIGURE 2.

Reconstitution of the supercomplexes from individual CIII and CIV in the presence of different lipid mixtures. A, BN-PAGE stained with Coomassie R-250 (left panel) and Western blot (right panel) with red or green fluorescent-tagged secondary antibodies detecting CIII or CIV, respectively, are shown. Samples were CIII (lanes 1), CIV (lanes 2), and CIII and CIV together (lanes 3), all in the absence of added phospholipid. B, BN-PAGE stained with Coomassie R-250 (left panel) and Western blot (2nd panel from left) with red or green fluorescent-tagged polyclonal antibodies directed against CIII or CIV, respectively, Western blot with polyclonal antibody against CIII (2nd panel from right), and Western blot with polyclonal antibody against CIV (right panel) are shown. Samples were CIII and CIV reconstituted in proteoliposomes containing an equimolar mixture of PC and PE supplemented with 20% CL (lanes 1) or native purified supercomplexes (lanes 2). The composition of the various bands is noted based on antibody cross-reactivity and mobility relative to the native trimer and tetramer. SC denotes supercomplex. C, BN-PAGE and Western blots as in B of CIII and CIV reconstituted together in liposomes containing an equimolar mixture of PC and PE (lanes 1) supplemented with 20% CL (lanes 2), PA 1% (lanes 3), PG 1% (lanes 4), PI 15% (lanes 5), and PS 2% (lanes 6) of the total phospholipid. D, same as in C, except all anionic phospholipids were supplemented to 20% of the total phospholipid. E, quantification of the different species visualized in the BN-PAGE after Coomassie R-250 staining (mean value of at least four different experiments with bars indicating standard deviation). In white is CIII; in light gray is the supercomplex trimer, and in dark gray is the supercomplex tetramer.

FIGURE 3.

CL concentration dependence for supercomplex formation in proteoliposomes. Quantification of the amount of tetramer (A) and trimer (B) obtained after reconstitution in the presence of an equimolar mixture of PC and PE containing variable amounts of CL. The results shown are the average of four different experiments, and the bars indicate standard deviation.

FIGURE 4.

Reconstitution of supercomplexes after phospholipase treatment of CIV. A, BN-PAGE stained with Coomassie R-250 and Western blot with CIII and CIV polyclonal antibodies of CIII and CIV with (+) or without (−) phospholipase treatment followed by reconstitution in either PC/PE liposomes (A) or PC/PE/CL liposomes (B). SC denotes supercomplex.

FIGURE 5.

In-gel activity of reconstituted CIII and CIV. CIII and CIV were reconstituted in the presence of PC, PE, and CL (CL 40%). For visualization of CIII activity, the digitonin-solubilized proteoliposome sample was subjected to colorless native-PAGE (A). For CIV activity, a BN-PAGE (B) was used. In-gel activities were performed as described under “Experimental Procedures” (lanes 1), or protein identification of the bands by Western blots (lanes 2) using antibody against CIII (A) or CIV (B) was used. SC denotes supercomplex.

FIGURE 6.

Single particle EM of reconstituted supercomplex and native III₂IV₂. After reconstitution of CIII and CIV in PC/PE/CL (CL 20%) liposomes, the III₂IV₂ supercomplex was purified by sucrose gradient centrifugation after solubilization with digitonin. BN-PAGE (A) of aliquots taken from the lower half of the sucrose gradient is shown. Fractions 2 and 3 near the bottom of the sucrose gradient were enriched in III₂IV₂ supercomplex and were used for the single particle EM analysis. Projection maps of the side view of the isolated reconstituted supercomplex tetramer stained with ammonium molybdate (B) and the purified natural supercomplex stained with ammonium molybdate (C) are shown. The number of particles used for each average is indicated on the top of each projection. Scale bar, 10 nm. SC denotes supercomplex.

FIGURE 7.

Presence of Rcf1p in the reconstituted supercomplexes. BN-PAGE stained with Coomassie R-250 and Western blots using CIV or Rcf1p polyclonal antibodies (as indicated) of the digitonin extracts containing supercomplexes reconstituted from CIII and CIV in the presence of PC and PE (lanes 1) or PC, PE, and CL (20% CL) (lanes 2) are shown. SC denotes supercomplex.