Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability.

Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

The role of protein quality control in mitochondrial protein homeostasis under oxidative stress.

Mitochondria contribute significantly to the cellular production of ROS. The deleterious effects of increased ROS levels have been implicated in a wide variety of pathological reactions. Apart from a direct detoxification of ROS molecules, protein quality control mechanisms are thought to protect protein functions in the presence of elevated ROS levels. The reactivities of molecular chaperones and proteases remove damaged polypeptides, maintaining enzyme activities, thereby contributing to cellular survival both under normal and stress conditions. We characterized the impact of oxidative stress on mitochondrial protein homeostasis by performing a proteomic analysis of isolated yeast mitochondria, determining the changes in protein abundance after ROS treatments. We identified a set of mitochondrial proteins as substrates of ROS-dependent proteolysis. Enzymes containing oxidation-sensitive prosthetic groups like iron/sulfur clusters represented major targets of stress-dependent degradation. We found that several proteins involved in ROS detoxification were also affected. We identified the ATP-dependent protease Pim1/LON as a major factor in the degradation of ROS-modified soluble polypeptides localized in the matrix compartment. As Pim1/LON expression was induced significantly under ROS treatment, we propose that this protease system performs a crucial protective function under oxidative stress conditions.

Chaperones and proteases--guardians of protein integrity in eukaryotic organelles.

Organelles like mitochondria, chloroplasts, or the endoplasmic reticulum are essential subcompartments of eukaryotic cells that fulfill important metabolic tasks. Organellar protein homeostasis is maintained by a combination of specific protein biogenesis processes and protein quality control (PQC) mechanisms that together guarantee the functional state of the organelle. According to their endosymbiontic origin, mitochondria and chloroplasts contain internal PQC systems that consist of a cooperative network of molecular chaperones and proteases. In contrast, the endoplasmic reticulum employs the main cytosolic degradation machinery, the proteasome, for the removal of damaged or misfolded proteins. Here we present and discuss recent experimental insights into the molecular mechanisms underlying organellar PQC processes.

Structure and function of Hsp78, the mitochondrial ClpB homolog.

The cellular role of Hsp100/Clp chaperones in maintaining protein stability is based on two functional aspects. Under normal growth conditions they represent components of cellular protein quality control machineries that selectively remove damaged or misfolded polypeptides in cooperation with specific proteases. After thermal stress, proteins of the ClpB subfamily have the unique ability to directly resolubilize aggregated polypeptides in concert with Hsp70-type chaperones, leading to the recovery of enzymatic activity. Hsp78, the homolog of the bacterial chaperone ClpB in mitochondria of eukaryotic organisms, participates in both protective activities. Hsp78 is involved in conferring thermotolerance to the mitochondrial compartment but also participates in protein degradation by the matrix protease Pim1. Despite the high sequence conservation between Hsp78 and ClpB, an analysis of the structural properties revealed significant differences. The identified mitochondrial Hsp78s do not contain N-terminal substrate-binding domains. In addition, formation of the oligomeric chaperone complex was more variable as anticipated from the studies with bacterial ClpB. Hsp78 predominantly formed a trimeric complex under in vivo conditions. Hence, mitochondrial Hsp78s form a distinct subgroup of the ClpB chaperone family, exhibiting specific structural and functional properties.