The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis

Eduard Hofsetz,Fatih Demir,Karolina Szczepanowska,Alexandra Kukat,Jayachandran N Kizhakkedathu,Aleksandra Trifunovic,Pitter F Huesgen

doi:10.1074/mcp.ra120.002082

Abstract

The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp-/- mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

Highlights

Candidate substrates identified by N termini accumulation and interaction with inactive ClpXP. UQCRC1, HSPA9 and ornithine aminotransferase (OAT) validated biochemically as high confidence ClpXP substrates
A total of 1558 N-terminal peptides from 322 proteins were identified after enrichment by Terminal Amine Isotope Labeling of Substrates (TAILS) (Fig. 1A, supplemental Table S2). 1058 N-terminal peptides were derived from 214 proteins with UniProt-annotated known or predicted mitochondrial localization (Fig. 1B, supplemental Table S3)
262 N-terminal peptides mapped to ”expected” positions, which we here define as N termini starting at positions 1 or 2 of the protein models or within 5 amino acids of known or predicted signal peptide (SP), mitochondrial targeting signal (MTS) or propeptide cleavage sites (Fig. 1D)

Summary

Graphical Abstract

We combined quantitative proteomics with N terminome profiling and substrate trapping AP-MS to identify new ClpXP substrates. Profiling of protein N termini in control and CLPP-deficient heart mitochondria by Terminal Amine Isotope Labeling of Substrates (TAILS) [27] revealed altered cleavages in accumulating candidate ClpXP substrates, including OAT and HSPA9, as well as moderate but widespread alterations in mitochondrial protein processing. We identified putative substrates by expressing FLAG-tagged, proteolytically active and inactive versions of CLPP in ClppϪ/Ϫ mouse embryonic fibroblasts (MEFs), followed by affinity purification and mass spectrometric quantification [9] Using this approach, we identified a small subset of trapped mitochondrial matrix proteins including UQCRC1, POLDIP2, and NDUFV2 as candidate substrates. Our results provide a first large-scale experimental characterization of protein maturation and processing in heart mitochondria and support the hypothesis that mammalian ClpXP primarily functions as a proteolytic regulator of specific substrates, and directly or indirectly contributes to mitochondrial protein quality control

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION