Identification of stress‐induced mitochondrial proteins in cultured tobacco cells

Abstract

Under stress conditions, small and large heat‐shock proteins cooperatively fulfil molecular chaperone activities within many cellular compartments, including mitochondria. Here, we report the identification and characterization of 10 newly synthesized low‐molecular‐weight heat stress‐induced proteins (SIPs) found solely in the mitochondrial fraction of suspension‐cultured cells of tobacco (Nicotiana tabacum L., strain Virginia Bright Italia‐0). These SIPs were shown to be encoded by the nuclear genome and none was detected in non‐stressed cells. Mass spectroscopy analysis revealed that eight SIPs belonged to the group of small heat‐shock proteins, whilst one protein shared significant homology with transcription factors. De novo synthesis of all SIPs was detected within 10 min of the commencement of heat stress and continued for the whole 12 h stress interval. Moreover, nine of the SIPs were stable for 24 h following stress termination. SIP1, which showed the most intense synthesis profile, was found to be phosphorylated. The most intense stress response was observed mainly during the exponential growth phase of the cell culture. Native electrophoresis of mitochondrial protein complexes revealed an association of all the SIPs in high‐molecular‐weight complexes with no free SIPs left in mitochondria. These complexes were localized to the mitochondrial membrane fraction.