Selective Transfer of Calcium from an Acidic Compartment to the Mitochondrion of Trypanosoma brucei: MEASUREMENTS WITH TARGETED AEQUORINS

Abstract

Organelle compartments are used by cells as reservoirs of exchangeable Ca2+ and as Ca2+ buffers. The following study uses recombinant aequorins (CYT-AEQ and MT-AEQ) to measure the dynamics of Ca2+ flux between organelles in procyclic forms of the pathogenic protozoan, Trypanosoma brucei. Emphasis is placed on the exchange between an acidic Ca2+ reservoir and the mitochondrion. The mammalian mitochondrial targeting sequence was functional in trypanosomes as determined by immunoblots, immunolocalizations, and the observation that MT-AEQ was in a compartment whose Ca2+ uptake was inhibited 82% with carbonyl cyanide p-trifluoromethoxyphenylhydrazone and KCN. The resting level of free calcium ion concentration in the mitochondrion ([Ca2+]mit) was slightly higher than that in the cytoplasm ([Ca2+]cyt) (400 +/- 50 nM and 290 +/- 40 nM, respectively). Melittin (125 nM) disrupted Ca2+ homeostasis by inducing Ca2+ influx across the plasma membrane. [Ca2+]cyt became slightly elevated to 410 +/- 100 nM, whereas [Ca2+]mit was selectively increased approximately 12-fold, with a broad peak at 4.8 +/- 1.9 microM. At the peak, the mitochondrion contained approximately three times more free Ca2+ than the cytosol. However, mitochondrial retention of the Ca2+ was transient. Similar selective transport into the mitochondrion was observed when Ca2+ efflux from an acidic compartment was induced with monensin (2 microg/ml) in the presence of 5 mM EGTA. [Ca2+]cyt was transiently elevated to 400 +/- 50 nM, whereas [Ca2+]mit was elevated to 3.3+/-1.3 microM. When cells were treated sequentially with monensin (2 microg/ml) and then melittin (200 nM), mitochondrial Ca2+ transport was normal. However, [Ca2+]cyt became elevated to a level that was 1.4-fold higher than with melittin alone. Overall, these data demonstrate that the trypanosome mitochondrion is not a reservoir of exchangeable Ca2+ in the resting cell. However, Ca2+ is selectively channeled to the mitochondrion from the plasma membrane or acidic Ca2+ storage compartment. Additionally, the acidic compartment contributes to maintenance of Ca2+ homeostasis in response to melittin.