The plant i-AAA protease controls the turnover of an essential mitochondrial protein import component

Abstract

Mitochondria are multifunctional organelles that play a central role in energy metabolism. Owing to the life-essential functions of these organelles, mitochondrial content, quality and dynamics are tightly controlled. Across the species, highly conserved ATP-dependent proteases prevent malfunction of mitochondria through versatile activities. This study focuses on a molecular function of the plant mitochondrial inner membrane-embedded AAA protease (denoted i-AAA) FTSH4, providing its first bona fide substrate. Here, we report that the abundance of the Tim17-2 protein, an essential component of the TIM17:23 translocase (Tim17-2 together with Tim50 and Tim23), is directly controlled by the proteolytic activity of FTSH4. Plants that are lacking functional FTSH4 protease are characterized by significantly enhanced capacity of preprotein import through the TIM17:23-dependent pathway. Taken together, with the observation that FTSH4 prevents accumulation of Tim17-2, our data point towards the role of this i-AAA protease in the regulation of mitochondrial biogenesis in plants.

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

“…Briefly, we utilized an Arabidopsis thaliana line that produces, instead of functional wild type FTSH4, a proteolytically inactivated tagged version of this protease ( ftsh4-1 FTSH4 TRAP.FLAG ). Proteolytic activity of FTSH4 was abolished by a single point mutation in the catalytic center of the proteolytic chamber [ 12 , 15 ]. Since AAA domain of the FTSH4 TRAP.FLAG mutant is active, the substrates are translocated into the proteolytic chamber, where they are not degraded and thus remain trapped [ 15 ].…”

“…We found an essential subunit of TIM17:23 translocase, Tim17-2, among proteins co-purifying with FTSH4 TRAP.FLAG . This protein was recently identified as a physiological substrate of the plant i -AAA protease [ 12 ]. FTSH4 protease regulates pre-protein influx into the mitochondria through the turnover of the Tim17-2 subunit.…”

“…We monitored the kinetics of degradation of candidate proteins inside mitochondria isolated from ftsh4 mutant and wild type plants. It is expected that the stability of proteolytic substrates will be enhanced in the mitochondria of mutants devoid of functional protease required for their turnover [ 12 , 33 ]. This assay allowed us to identify two novel FTSH4 proteolytic substrates, such as the component of mitochondrial protein import machinery Pam18-2 and the processed form of mitochondrial pyruvate carrier (MPC4).…”

“…Arabidopsis thaliana lines: ecotype Col-0 wild type, T-DNA insertion lines ftsh4-1 (SALK_035107/TAIR) [ 35 ] and ftsh4-1 FTSH4 TRAP.FLAG ( ftsh4-1 FTSH4(H486Y)) mutant line [ 12 ], were grown on 0.5 Murashige and Skoog (MS) medium supplemented with 3% ( w / v ) sucrose in chambers in a 16 h light/8 h dark (long day, LD) photoperiod at 22 °C for 2 weeks with a light intensity of 150 μmol m −2 s −1 [ 12 ].…”

“…Thus far, only one physiological substrate of FTSH4 has been described. FTSH4 is required for the turnover of the essential subunit of the TIM23 translocase, Tim17-2, indicating FTSH4-dependent proteolytic regulation of pre-protein influx into the plant mitochondria [ 12 ]. However, under stress conditions, loss of FTSH4 is linked to broad pleiotropic phenotypes including oxidative stress, imbalance in phospholipid metabolism, perturbation in oxidative phosphorylation system activity and aberrant mitochondrial morphology, suggesting that plant i -AAA protease, like its mammalian homologue, controls distinct processes inside the mitochondria [ 13 , 14 ].…”