Ca2+ transport in mitochondria from yeast expressing recombinant aequorin.
We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca(2+) transport in vivo and in vitro. When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl(2), the matrix free Ca(2+) concentration is 200 nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca(2+) and promptly increases the value to 1.2 microM. Elevated Ca(2+) concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca(2+) when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca(2+) release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502-40509]. When a respiratory substrate is not present, Ca(2+) enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca(2+) and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K(1/2) value for Ca(2+) that is near to 900 nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca(2+) in yeast mitochondria is a function of the matrix Ca(2+) content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca(2+) transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca(2+) in yeast.
Jung
Dennis W
DW
Department of Molecular and Cellular Biochemistry, The Ohio State University Medical Center, Columbus, OH 43210, USA.
Bradshaw
Patrick C
PC
Litsky
Monica
M
Pfeiffer
Douglas R
DR
D000331:Aequorin / Q000096:biosynthesis*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D027682:Cation Transport Proteins; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000648:ultrastructure*
D027682:Cation Transport Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D000331:Aequorin; D002118:Calcium