title: Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria.
abstract: The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.
authors.affiliation: Department of Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht, NL-3584 CH, The Netherlands.
authors.forename: Marjolein J F W
authors.initials: MJ
authors.lastname: Janssen
authors.forename: Ben
authors.initials: B
authors.lastname: de Kruijff
authors.forename: Anton I P M
authors.initials: AI
authors.lastname: de Kroon
mesh_terms: D004734:Energy Metabolism / Q000502:physiology; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000502:physiology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome); D008274:Magnesium / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D026901:Membrane Transport Proteins / Q000378:metabolism; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins; D010101:Oxygen Consumption / Q000502:physiology*; D010539:Permeability; D010710:Phosphates / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014357:Trypsin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D010710:Phosphates; D029701:Saccharomyces cerevisiae Proteins; C066555:Tom40 protein, S cerevisiae; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D014357:Trypsin; D008274:Magnesium