Calcium depletion and calmodulin inhibition affect the import of nuclear‐encoded proteins into plant mitochondria

Abstract

SUMMARYMany metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/ calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.

The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively

“…Very little is known about the extent of CaM mediated calcium regulation in different plant organelles but evidence for calcium/calmodulin regulation of chloroplast, mitochondrial and peroxisomal functions has been described Chigri et al 2005;Jarrett et al 1982;Kuhn et al 2009;Miernyk et al 1987;Pou De Crescenzo et al 2001;Sauer and Robinson 1985;Yang and Poovaiah 2002). In peroxisomes, CaM has been associated with catalase activity (Yang and Poovaiah 2002).…”

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

“…Furthermore, the EF-hand protein LETM1 and the calmodulin-like protein CML30 have recently been found to be localized in mitochondria, and additional proteins with Ca 2+ binding motifs have been detected in proteomic data sets of plant mitochondria (Chigri et al, 2012;Zhang et al, 2012;Salvato et al, 2014). Moreover, there is evidence for Ca 2+ dependence of mitochondrial permeability transition, protein import, and respiration in plants (Miernyk et al, 1987;Arpagaus et al, 2002;Kuhn et al, 2009;Manzoor et al, 2012). Nevertheless, it remains unclear if metabolic flux through pyruvate dehydrogenase and the tricarboxylic acid cycle can be regulated by Ca 2+ in vivo (Møller and Rasmusson, 1998).…”

Novel Role of Calmodulin in Regulating Protein Transport to Mitochondria in a Unicellular Eukaryote

“…The involvement of Ca 2ϩ /CaM in translocation of a mitochondrial protein, alternative oxidase of Pisum sativum, has been shown through Ca 2ϩ depletion studies (using A23187 and ionomycin) and use of CaM inhibitor ophiobolin A. However, the study did not project either CaM binding sites or any CaM interaction with the protein (40), leaving open the possibilities of involvement of other components influenced by ionophores and inhibitors.…”