Energy-Linked Ultrastructural Transformations in Isolated Liver Mitochondria and Mitoplasts

132

103

The coupling between molecular diffusion and the structure and function of the rat liver mitochondrial matrix was explored using fluorescence anisotropy techniques and electron microscopy. The results confirm that matrix ultrastructure and the concentration of matrix protein are influenced by the respiratory state of mitochondria and the osmolarity of the external medium. At physiological osmolarity, a fluorescent metabolite-sized probe was found to diffuse slowly in the mitochondrial matrix but not to be completely immobile. In addition, significant differences in diffusion rates were found to exist between different mitochondrial respiratory states, with the slowest diffusion occurring in states with the highest matrix protein concentration. These data support the concept of a matrix structure in which diffusion is considerably hindered due to limited probe-accessible water and further suggest that volume-dependent regulation of matrix protein packing may modulate metabolite diffusion and, in turn, mitochondrial metabolism.

Section: Resultsmentioning

confidence: 99%

Dynamics, structure, and function are coupled in the mitochondrial matrix.

Scalettar

Abney

Hackenbrock

1991

132

103

“…Subsequent removal of the outer membrane and purification of the inner membrane/matrix (mitoplast) fraction was carried out by use of a controlled digitonin incubation (9,10). The topographically complex inner membrane was converted to a simple spherical configuration by two washes in the isolation medium diluted 1:7.5 (40 mosM) (9).…”

Section: Methodsmentioning

confidence: 99%

Fusion of liposomes with mitochondrial inner membranes

Schneider

Lemasters

Höchli

et al. 1980

Self Cite

A procedure is outlined for the fusion of mixed phospholipid liposomes (small unilamellar vesicles) with the mitochondrial inner membrane, which enriches the membrane lipid bilayer 30-700% in a controlled fashion. Fusion was initiated by manipulation of the pH of a mixture of freshly sonicated liposomes and the functional inner membrane/matrix fraction of rat liver mitochondria. During the pH fusion procedure, liposomes became closely apposed with and sequestered by the inner membranes as revealed by freeze-fracture electron microscopy. After the pH fusion procedure, a number of ultrastructural, compositional, and functional characteristics were found to be proportionally related: the membrane surface area increased; the lateral density distribution of intramembrane particles (integral proteins) in the plane of the membrane decreased whereas the particles remained random; the membrane became more buoyant; the ratio of membrane lipid phosphorus to total membrane protein increased; the ratio of membrane lipid phosphorus to heme a of cytochrome c oxidase increased; and the rate of electron transfer between some interacting membrane oxidoreduction proteins decreased. These data reveal that liposomal phospholipid was incorporated into the membrane bilayer (not simply adsorbed to the membrane surface) and that integral membrane proteins diffused freely into the laterally expanding bilayer. Furthermore, the data suggest that the rate of electron transfer may be limited by the rate of lateral diffusion of oxidoreduction components in the bilayer of the mitochondrial inner membrane.Although a number of advances have been reported regarding the theoretical aspects of membrane fusion generally and the fusion of liposomes with plasma membranes specifically (1-5), the evidence for liposome-membrane fusion appears to be more circumstantial than unequivocal (5). The essential difficulty in the assessment of fusion has been in the quantitation of the liposomal lipid fused with and incorporated into the membrane bilayer.Recent observations in our laboratory on the lateral diffusion of integral proteins generally (6) and of cytochrome c oxidase specifically (7) in the mitochondrial energy-transducing membrane prompted us to develop a liposome-membrane fusion procedure that can be utilized to increase the surface area of the lipid bilayer of the functional membrane in a quantitatively controlled fashion. The need for such an approach has grown out of our interest in ascertaining the effects of the physical properties of the membrane bilayer lipid on diffusional, collisional, and catalytic events that occur among the membrane oxidoreduction proteins and that are fundamental to the kinetics and mechanisms of electron transfer and oxidative phosphorylation (8).In the present report we outline a simple fusion procedure for incorporating limited to massive quantities of liposomal lipids into the mitochondrial energy transducing membrane and describe quantitative assessment of several ultrastructural, compositional, and functio...

“…Subsequent removal of the outer membrane and purification of the inner membrane/matrix (mitoplast) fraction was carried out by use of a controlled digitonin incubation (7,8). The complex inner membrane was converted to a simple spherical, functional membrane by washing and resuspending in a 1:7.5 diluted (40 mosM) albumin-free H medium as described (7,9) (medium is designated H40 medium). Preparation and analysis of affinity-purified rabbit immunoglobulin monospecific for cytochrome c oxidase, affinity-purified goat anti-rabbit immunoglobulin, and ferritin-conjugated immunoglobulin were as described (6).…”

Section: Methodsmentioning

confidence: 99%

Lateral translational diffusion of cytochrome c oxidase in the mitochondrial energy-transducing membrane

Höchli

Hackenbrock

1979

Self Cite

The degree of freedom for lateral translational diffusion by cytochrome c oxidase and other integral proteins in the energ-transducing membrane of the mitochondrion was determined by combining the use of an immunoglobulin probe monospecific for the oxidase with thermotropic lipid phase transitions. Lateral mobility of the oxidase was monitored by observing the distribution of the immunoglobulin probe on the membrane surface by deep-etch electron microscopy and by observing the distribution of intramembrane particles (integral proteins) in the hydrophobic interior of the membrane by freeze-fracture electron microscopy. Incubation of the membrane with the immunoglobulin resulted in a time-dependent clustering of predominantly large intramembrane particles. Low temperature-induced lipid phase transitions resulted in the close packing of all intramembrane particles and cytochrome c oxidase by lateral exclusion from domains of gel-state bilayer lipid and was completely reversible. However, when cytochrome c oxidase was crosslinked through an immunoglobulin lattice prior to returning the membrane to above the lipid phase transition temperature, small intramembrane particles rerandomized while the large oxidase-related particles remained clustered. These observations reveal that cytochrome c oxidase can diffuse laterally in the energy-transducing membrane, either independently of all other integral proteins or in physical union with one or more other integral proteins. In addition, many other as yet unidentified smaller integral proteins can diffuse independently of the oxidase. Whether or not proteins integral to the mitochondrial energy-transducing membrane can undergo two-dimensional lateral diffusion independently of one another relates directly to the molecular events implicit in the mechanisms of electron transfer and energy coupling in oxidative phosphorylation (1). Electron transfer between the respiratory proteins as well as energy coupling between these proteins and the oligomycinsensitive ATPase may occur through direct physical contact, either through a relatively permanent protein-protein complex or by alternating protein-protein collisions. Such direct physical contact is required in coupling mechanisms in which the energy derived from electron transfer is transduced through covalent high-energy bonds or through energized protein conformational states to generate the synthesis of ATP (2, 3). Alternatively, no physical contact, permanent or alternating, is required in coupling mechanisms in which the energy derived from electron transfer is transduced through a proton gradient across or in the mitochondrial membrane to generate the synthesis of ATP (4, 5).Cytochrome c oxidase is one of 30 or so proteins occurring in the energy-transducing membrane and participates in electron-transfer and energy-coupling processes. The oxidase is a completely transmembranous integral protein (6). In the present communication we report on the application of a metabolic and visual immunoglobulin probe monospecific f...