Global Analysis of the Mitochondrial N-Proteome Identifies a Processing Peptidase Critical for Protein Stability

Abstract

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.

A Phosphodiesterase 2A Isoform Localized to Mitochondria Regulates Respiration

“…Thus, alternative splicing of PDE2A directs distinct subcellular localization, enabling assembly of independent, localized cNMP signaling domains. During import, PDE2A2 might be proteolytically processed, as suggested by a putative processing site RRG-QQ and the resulting N-terminal Gln, which is the third most abundant N-terminal residue resulting from mitochondrial processing (44). Consistent with the N terminus being more flexible, as typically observed for localization sequences, the PDE2A fragment whose structure was recently solved contained the whole protein except for the N terminus (54).…”

“…The N Terminus of PDE2A Isoform 2 Promotes Mitochondrial Localization-Mitochondrial transport systems can import proteins that contain different types of localization signals, but most matrix proteins contain an N-terminal sorting signal, which is often proteolytically removed during import (43,44). Three PDE2A isoforms have been described, PDE2A1, PDE2A2, and PDE2A3, which are encoded by a single mammalian PDE2A gene and differ in their N-terminal sequences due to alternative splicing (22)(23)(24).…”

Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import

“…Regardless of the mechanism, generation of alternative mature forms via differential, two-step processing may be a general mechanism to allow production of multiple forms of mitochondrial matrix proteins destined to perform different functions. This is especially worth considering given the large number of mitochondrial proteins subjected to cleavage upon import based on yeast proteomic studies (24). Incidentally, these studies show that both the putative yeast ortholog of MRPL12 (YGL068W) and MRPL10 are cleaved upon import and that the latter is a substrate of a yeast MIP (24).…”

“…This is especially worth considering given the large number of mitochondrial proteins subjected to cleavage upon import based on yeast proteomic studies (24). Incidentally, these studies show that both the putative yeast ortholog of MRPL12 (YGL068W) and MRPL10 are cleaved upon import and that the latter is a substrate of a yeast MIP (24).…”

“…The Two Forms of MRPL12 Are Generated by Differential Two-step Proteolytic Cleavage-Many nucleus-encoded, mitochondria-targeted proteins are processed by proteolytic cleavage of the mitochondrial-targeting sequence during import by the mitochondrial processing protease (MPP) (24). A subset of these is subsequently cleaved by the mitochondrial intermediate protease (MIP).…”