Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

Abstract

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4's in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4's physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

Keywords: AAA protease; ATP-dependent proteolysis; carbonylated proteins; inner mitochondrial membrane proteostasis; mitochondria.

Figure 1

FTSH4 substrate trapping assay (A) Overview of FTSH4 substrate-trapping assay. Cartoon illustrating the experimental workflow; (B) Eluted fractions resolved on SDS-PAGE and stained with CBB. IMS—intermembrane space.

Figure 2

Immunoblot analysis of FTSH4 substrate trapping assay samples. Mitochondria from control (ftsh4-1) and ftsh4-1 FTSH4^TRAP.FLAG were solubilized with digitonin and subjected to immunoprecipitation with anti-FLAG affinity matrix. The precipitated proteins were immunoblotted with antibodies against the indicated proteins. IN—input (5%), FT—flow-through (5%), W—Wash, E—eluate (50%).

Figure 3

Novel proteolytic substrates of FTSH4 protease. (A) Degradation of Pam18-2 by FTSH4 following its in vitro import into mitochondria. Radiolabeled Pam18-2 was imported into mitochondria derived from either wild type or ftsh4-1 plants. The stability of newly imported precursor upon further incubation at 26 °C was analyzed by SDS-PAGE and autoradiography. Quantification of [³⁵S] Pam18-2 in mitochondria is represented in the lower panel. Newly imported Pam18-2 was set to 100%. Data represent mean ± SD of three independent experiments. * p < 0.02 and ** p < 0.003 (t-student test); (B) Degradation of MPC4 processed form by FTSH4 following its in vitro import into mitochondria. Radiolabeled MPC4 was imported into mitochondria derived from either wild type or ftsh4-1 plants. The stability of newly imported MPC4 upon further incubation at 26 °C was analyzed by SDS-PAGE and autoradiography. Quantification of [³⁵S] MPC4 processed form in mitochondria is represented in the lower panel. Newly imported MPC4 was set to 100%. Data represent mean ± SD of three independent experiments. * p < 0.05 (t-student test). m—MPC4 processed form, p—MPC4 precursor.

Figure 4

FTSH4 degrades oxidatively damaged mitochondrial proteins. (A) Anti-DNP (dinitrophenyl hydrazone) immunoblot detection of carbonylated proteins co-precipitating with FTSH4^TRAP.FLAG. The cross-reaction of IgG light chain is marked with asterisk. (B) The degradation of mitochondrial carbonylated proteins by FTSH4 protease was assessed by immunoblot analysis of the levels of carbonylated proteins in mitochondrial extract from ftsh4-1 mutant incubated at 35 °C with or without FTSH4 protein. (C) Quantification of the remaining carbonylated proteins after 2 h incubation with or without FTSH4 protein, shown in (B). For each sample, the amount of carbonylated proteins was set to 100% at time point 0 h. Data represent mean ± SEM of three independent experiments. ** p < 0.03 (t-student test).