Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis.
Mitochondrial F(1)F(o)-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F(1)F(o)-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F(1)F(o)-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F(1)F(o)-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F(1) catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F(1) to the lipid monolayer and the F(o) membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F(o) by electron microscopy and AFM.
Arechaga
Ignacio
I
MRC Dunn Human Nutrition Unit, Hills Road, Cambridge, UK. arechagai@unican.es <arechagai@unican.es>
Fotiadis
Dimitrios
D
D000818:Animals; D002417:Cattle; D003460:Crystallization; D006639:Histidine / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D008051:Lipid Bilayers; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology; D011487:Protein Conformation; D021122:Protein Subunits; D011510:Proteolipids / Q000737:chemistry* / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
D008051:Lipid Bilayers; D021122:Protein Subunits; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; C033223:polyhistidine; D006639:Histidine; D006180:Proton-Translocating ATPases