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

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.

Mitochondrial phosphatase PTPMT1 is essential for cardiolipin biosynthesis.

PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1 deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.

The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis.

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.

Phosphatidylethanolamine and monoglucosyldiacylglycerol are interchangeable in supporting topogenesis and function of the polytopic membrane protein lactose permease.

To determine the specific role lipids play in membrane protein topogenesis in vivo, the orientation with respect to the membrane bilayer of Escherichia coli lactose permease (LacY) transmembrane (TM) domains and their flanking extramembrane domains was compared after assembly in native membranes and membranes with genetically modified lipid content using the substituted cysteine accessibility method for determining TM domain mapping. LacY assembled in the absence of the major membrane lipid phosphatidylethanolamine (PE) does not carry out uphill transport of substrate and displays an inverted orientation for the N-terminal six-TM domain helical bundle (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). Strikingly, the replacement of PE in vivo by the foreign lipid monoglucosyldiacylglycerol (MGlcDAG), synthesized by the Acholeplasma laidlawii MGlcDAG synthase, restored uphill transport and supported the wild type TM topology of the N-terminal helical bundle of LacY. An interchangeable role in defining membrane protein TM domain orientation and supporting function is played by the two most abundant lipids, PE and MGlcDAG, in gram-negative and gram-positive bacteria, respectively. Therefore, these structurally diverse lipids endow the membrane with similar properties necessary for the proper organization of protein domains in LacY that are highly sensitive to lipids as topological determinants.

Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli.

The mechanisms by which lipid bilayer properties govern or influence membrane protein functions are little understood, but a liquid-crystalline state and the presence of anionic and nonbilayer (NB)-prone lipids seem important. An Escherichia coli mutant lacking the major membrane lipid phosphatidylethanolamine (NB-prone) requires divalent cations for viability and cell integrity and is impaired in several membrane functions that are corrected by introduction of the "foreign" NB-prone neutral glycolipid alpha-monoglucosyldiacylglycerol (MGlcDAG) synthesized by the MGlcDAG synthase from Acholeplasma laidlawii. Dependence on Mg(2+) was reduced, and cellular yields and division malfunction were greatly improved. The increased passive membrane permeability of the mutant was not abolished, but protein-mediated osmotic stress adaptation to salts and sucrose was recovered by the presence of MGlcDAG. MGlcDAG also restored tryptophan prototrophy and active transport function of lactose permease, both critically dependent on phosphatidylethanolamine. Three mechanisms can explain the observed effects: NB-prone MGlcDAG improves the quenched lateral pressure profile across the bilayer; neutral MGlcDAG dilutes the high anionic lipid surface charge; MGlcDAG provides a neutral lipid that can hydrogen bond and/or partially ionize. The reduced dependence on Mg(2+) and lack of correction by high monovalent salts strongly support the essential nature of the NB properties of MGlcDAG.