Abstract
The accumulation and retention of Ca(2+) by yeast mitochondria (Saccharomyces cerevisiae) mediated by ionophore ETH 129 occurs with a variable efficiency in different preparations. Ineffective Ca(2+) transport and a depressed membrane potential occur in parallel, are exacerbated in parallel by exogenous free fatty acids, and are corrected in parallel by the addition of bovine serum albumin. Bovine serum albumin is not required to develop a high membrane potential when either Ca(2+) or ETH 129 are absent, and when both are present membrane potential is restored by the addition of EGTA in a concentration-dependent manner. Respiration and swelling data indicate that the permeability transition pore does not open in yeast mitochondria that are treated with Ca(2+) and ETH 129, whereas fatty acid concentration studies and the inaction of carboxyatractyloside indicate that fatty acid-derived uncoupling does not underlie the other observations. It is concluded that yeast mitochondria contain a previously unrecognized Ca(2+):2H(+) antiporter that is highly active in the presence of free fatty acids and leads to a futile cycle of Ca(2+) accumulation and release when exogenous Ca(2+) and ETH 129 are available. It is also shown that isolated yeast mitochondria degrade their phospholipids at a relatively rapid rate. The activity responsible is also previously unrecognized. It is Ca(2+)-independent, little affected by the presence or absence of a respiratory substrate, and leads to the hydrolysis of ester linkages at both the sn-1 and sn-2 positions of the glycerophospholipids. The products of this activity, through their actions on the antiporter, explain the variable behavior of yeast mitochondria treated with Ca(2+) plus ETH 129.
Highlights
In simpler eucaryotes such as yeast, the cell signaling roles of Ca2ϩ are less developed, and there is a corresponding reduction in the role of mitochondria in maintaining Ca2ϩ homeostasis
This is despite the fact that ETH 129 is a well behaved ionophore for Ca2ϩ in model systems, displaying properties that are highly reproducible [26], and despite the fact that the Ca2ϩ transport activities of mammalian mitochondria do not show this degree of variability as long as the permeability transition is avoided
Yeast Mitochondria Contain an Endogenous Ca2ϩ Transport Activity—Mammalian mitochondria that are treated with Ca2ϩ plus A23187 or ionomycin behave in much the same way as yeast mitochondria that are treated with Ca2ϩ plus ETH 129
Summary
Preparation of Yeast Mitochondria—The yeast strain W303–1A (S. cerevisiae) was grown aerobically at 30 °C in a medium containing 2% lactate, 1% yeast extract, 2% peptone, 0.05% dextrose, and 0.01% adenine at pH 5.0, and cells were harvested during the logarithmic phase (A600 ϭ 1.8 –2.2). The resulting preparations were maintained on ice and were suspended in 0.6 M mannitol, 20 mM HEPES (Kϩ) (pH 6.8), containing 0.75 mg/ml BSA2 and 0.1 mM EGTA. Determination of Ion Transport, ⌬⌿, and Related Parameters—Unless noted otherwise, mitochondria were incubated at 1 mg protein/ml and at ϳ25 °C in a medium that contained 0.6 M mannitol, 10 mM Pi (Kϩ), 10 mM HEPES (TEAϩ), pH 7.2, plus 1 mM ethanol as an oxidizable substrate. Determination of Free Fatty Acids and Mitochondrial Phospholipids—Samples containing 3 mg of mitochondrial protein were extracted by a modified Folch technique [19], following the addition of a known amount of heptadecanoic acid (17:0), which was employed as an internal standard [20]. Total mitochondrial phospholipids were estimated from measurements of lipid phosphorous in an aliquot of the initial extract [22]
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