Our research is directed at understanding the role phospholipids play in organization of membrane proteins into macro-molecular machines. This assumes membrane lateral heterogeneity and formation of a network of membrane lipid-protein domains. We are using two models, the highly specialized membrane of yeast mitochondria, containing enzymes of the oxidative phosphorylation system, and the multifunctional bacterial membrane of Escherichia coli. The bacterial model is employed to study interaction of phospholipids with the cytoskeletal proteins of the bacterial division machinery. We investigate the mutual influence of amphitropic cell division proteins, which undergo dynamic oligomerization on the membrane surface, and membrane phospholipids, in the formation of dynamic lipid-protein domains. Our hypothesis is that the domains enriched by the anionic phospholipid cardiolipin (CL) play an important role in the bacterial cell division process. We also participate in a collaborative study to explore the action of a number of antimicrobial agents, the primary target of which is anionic phospholipids. Using the yeast model we investigate the role of CL in organization of electron transfer enzymes of the respiratory chain into supra-molecular structures termed as supercomplexes. Although the existence of respiratory supercomplexes is now generally accepted, the factors that regulate their formation and function are not well understood. Earlier, using a yeast model system, we discovered a direct correlation between CL levels and levels of respiratory supercomplexes thus showing that CL played a central role in supercomplex formation. Subsequently, a significant amount of data have been accumulated by different groups demonstrating that in many diseases, including Barth syndrome, neurodegenerative diseases, heart failure, and cancer, the defects in respiratory supercomplex formation are associated with reduced levels of CL or alterations in the landscape of CL species. Currently we focus on understanding the precise molecular mechanism for the CL-dependent formation and stabilization of the supercomplexes. Our approach combines biochemical, structural and computational studies. We have performed the first in vitro reconstitution of the yeast supercomplex composed of Complex III and two Complexes IV from the purified individual complexes in CL containing liposomes. In our recent structural studies a 3D density map of the yeast respiratory supercomplex composed of Complex III and two Complexes IV was obtained by electron cryo-microscopy and image processing. The structure shows gaps between the transmembrane-localized interfaces of individual complexes consistent with the large excess of CL in the supercomplex over tightly bound CL in the structure of individual respiratory complexes. We hypothesize that non-integral CL molecules play an important role in supercomplex formation and may be involved in regulation of its stability under metabolic conditions where CL levels fluctuate.
Pseudo-atomic model of the IV1-III2-IV1 respiratory supercomplex from Saccharomyces cerevisiae constructed by docking of available crystal structures of Complexes III and IV into the 3D density map of the supercomplex (Mileykovskaya et al. 2012). Side or transmembrane view where the upper part is facing the mitochondrial matrix (MA) and the lower part is facing the inter-membrane space (IMS). Horizontal lines and shading indicate the position of phospholipid membrane bilayer (MEM). Complex III: cytochrome b, yellow, positions of bL low potential and bH high potential hemes (red) are indicated; Rieske protein, cornflower blue, FeS cluster, purple; cytochrome c1, cyan, heme (red) is indicated; Qcr6p, orange (marked by orange arrow); CL, dark blue; cytochrome c, pink with heme c in red. All other subunits are in light purple. Complex IV: subunit I (Cox1p, yeast), light blue; subunit II (Cox2p, yeast), sandy brown; subunit Cox5ap (marked by green arrow), position of CuA, hemes a (red) and a3 (red) are indicated. All other subunits of CIV are in olive green. S. cerevisiae CIII (PDB ID: 3CX5) was used, antibody fragment chains were removed. Bovine CIV (PDB ID: 1OCC), a monomer of the dimeric bovine CIV was used with chains K and M, which are not present in yeast, removed.
Mileykovskaya E., Dowhan W. “Visualization of phospholipid domains in Escherichia coli by using cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange”, J. Bacteriol. 182:1172-1175, 2000.
Mileykovskaya E., Dowhan W., Birke R.L., Zheng D., Lutterodt L.,Haines T.H. “Cardiolipin binds nonyl acridine orange by aggregating the dye at exposed hydrophobic domains on bilayer surfaces”, FEBS Lett. 507: 187-190, 2001.
Zhang M., Mileykovskaya E., Dowhan, W. “Gluing the Respiratory Chain Together: Cardiolipin Facilitates Supercomplex Formation in the Inner Mitochondrial Membrane”, J. Biol. Chem. 277: 43553-43556, 2002.
Mileykovskaya E., Fishov I., Fu X., Corbin B.D., Margolin W., Dowhan W. “Effect of phospholipid composition on MinD-membrane interactions in vitro and in vivo”, J. Biol. Chem. 278: 22193-22198, 2003.
Norris V., Woldringh C., Mileykovskaya E. “A hypothesis to explain division site selection in Escherichia coli by combining nucleoid occlusion and Min”, FEBS Lett. 561: 3-10, 2004.
Dowhan, W., Mileykovskaya, E., and Bogdanov, M. “Diversity and Versatility of Lipid-Protein Interactions Revealed by Molecular Genetic Approaches”, Biophys. Biochim. Acta 1666:19-39, 2004.
Zhang M., Mileykovskaya E., Dowhan W. “Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria”, J. Biol. Chem. 280: 29403-29408, 2005.
Mileykovskaya, E., Zhang, M., and Dowhan, W. “Cardiolipin in Energy Transducing Membranes”, Biochemistry (Moscow) 70:154-158, 2005.
Mileykovskaya, E. and Dowhan, W. “Role of membrane lipids in bacterial division-site selection”, Curr. Opin. Microbiol. 8:135-142, 2005.
Mileykovskaya E. “Subcellular localization of Escherichia coli osmosensory transporter ProP: focus on cardiolipin membrane domains”, Mol. Microbiol. 64:1419-1422, 2007.
Bogdanov, M., Mileykovskaya E. and Dowhan, W. “Lipids in the Assembly of Membrane Proteins and Organization of Protein Supercomplexes: Implications for Lipid-linked Disorders” in Lipids in Health and Disease; Series: Subcellular Biochemistry 49: 197-239, 2008.
Mazor, S., Regev, T., Mileykovskaya E., Margolin, W., Dowhan, W., Fishov, I. “Mutual effects of MinD-membrane interaction: I. Changes in the membrane properties induced by MinD binding”, Biochem. Biophys. Acta 1778: 2496-2505, 2008.
Mileykovskaya E. and Dowhan W., “Cardiolipin membrane domains in prokaryotes and eukaryotes”, Biochim. Biophys. Acta 1788: 2084-2091, 2009.
Mileykovskaya E. and Margolin W., Chapter 5-1: “Cell division”. Pp 149-177 in “Escherichia coli and Bacillus subtilis; the frontiers of molecular microbiology revised”, Editors: Yoshito Sadaie and Kouji Matsumoto, Research Signpost, Kerala, India, 2012.
Mileykovskaya E., Penczek P.A., Fang J., Mallampalli V.K., Sparagna G.C., Dowhan W. “Arrangement of the respiratory chain complexes in Saccharomyces cerevisiae supercomplex III2IV2 revealed by single particle cryo-electron microscopy”, J. Biol. Chem. 287: 23095-23103, 2012.
Tran T.T., Panesso D., Mishra N.N., Mileykovskaya E., Guan Z., Munita J.M., Reyes J., Diaz L., Weinstock G.M., Murray B.E., Shamoo Y., Dowhan W., Bayer A.S., Arias C.A.“Daptomycin-resistant Enterococcus faecalis diverts the antibiotic molecule from the division septum and remodels cell membrane phospholipids” MBio., 4(4). pii: e00281-13, 2013.
Bazan S., Mileykovskaya E., Mallampalli V.K., Heacock P., Sparagna G.C., Dowhan W. “Cardiolipin-dependent reconstitution of respiratory supercomplexes from purified Saccharomyces cerevisiae complexes III and IV”, J. Biol. Chem. 288: 401-411, 2013
Mileykovskaya E., Dowhan, W. “Cardiolipin-Dependent Formation of Mitochondrial Respiratory Supercomplexes” Chem. Phys. Lipids 2013 in press.
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Ph.D. in Biochemistry - Department of Bioenergetics A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Russia
Postdoctoral Research Fellow - A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia
Supramolecular organization of membrane protein complexes. Role of cardiolipin.,