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

Abstract

Lower eukaryotes like the kinetoplastid parasites are good models to study evolution of cellular pathways during steps to eukaryogenesis. In this study, a kinetoplastid parasite, Leishmania donovani, was used to understand the process of mitochondrial translocation of a nucleus-encoded mitochondrial protein, the mitochondrial tryparedoxin peroxidase (mTXNPx). We report the presence of an N-terminal cleavable mitochondrial targeting signal (MTS) validated through deletion and grafting experiments. We also establish a novel finding of calmodulin (CaM) binding to the MTS of mTXNPx through specific residues. Mutation of CaM binding residues, keeping intact the residues involved in mitochondrial targeting and biochemical inhibition of CaM activity both in vitro and in vivo, prevented mitochondrial translocation. Through reconstituted import assays, we demonstrate obstruction of mitochondrial translocation either in the absence of CaM or Ca 2؉ or in the presence of CaM inhibitors. We also demonstrate the prevention of temperature-driven mTXNPx aggregation in the presence of CaM. These findings establish the idea that CaM is required for the transport of the protein to mitochondria through maintenance of translocation competence posttranslation.

Sestrin2 facilitates glutamine‐dependent transcription of PGC‐1α and survival of liver cancer cells under glucose limitation

“…Whole cell lysates were prepared using 1× sample buffer containing 2% SDS, 10% glycerol, 0.1% 2‐mercaptoethanol, and 100 m m Tris/HCl (pH 6.8) and estimation of proteins was done using a CBX assay kit. Equal amount of protein samples were loaded and resolved on 12% SDS/PAGE gel and subsequently transferred onto nitrocellulose membrane as described previously . Furthermore, transblotted proteins were blocked using 5% BSA and immunoblotting was done using relevant primary and secondary antibodies.…”

Mitochondrial Dynamics and Heart Failure

“…The outer membrane is a selectively permeable membrane, containing integral membrane proteins and pores for transporting molecules. Proteins larger than 5000 Daltons require specific signaling sequences, mitochondria targeting sequence (MTS), to be transported across the outer membrane (2;49;60;192). A consequence of this is that the proteins found in this space differ significantly from the proteins found in the cytosol.…”

Elevated ergosterol protectsLeishmaniaparasites against antimony‐generated stress

“…They have a digenetic life cycle surviving alternatively within the insect and the mammalian host where they are exposed to a large amount of ROS. As a defense to host‐generated antileishmanial activity, these parasites express a number of defensive enzymes like the cytosolic and the mitochondrial form of the tryparedoxin peroxidases (6, 7), antioxidant thiols like trypanothione and ovothiol, but lack catalase and the selenium‐dependent glutathione peroxidases, which are the primary arsenal against the ROS and its products (8, 9). Nonetheless, these parasites are able to defend themselves well, create successful infections, and resist drugs.…”