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

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 mitochondrial N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA

“…These potential ClpP substrates were assessed on the STRING web platform, revealing a strongly significant protein–protein interaction enrichment ( p < 1 × 10 −16 ). STRING generated a diagram of protein–protein interactions that highlights a prominent network of mitoribosomal components and molecular chaperones, as described [ 12 , 16 ]; in comparison, the assembly factors for iron–sulfur cluster biosynthesis and respiratory chain elements as well as mitochondrial enzymes played a minor role ( Figure S1 ). The detailed analysis of enrichments ( Table S2 ) also documented the previously reported increase in cytosolic innate immunity mediators [ 15 ], and a novel elevation in secreted collagen precursors.…”

“…A wider spectrum of targets was described for the mitochondrial matrix peptidase ClpP, which plays important roles during the unfolded protein response for mitochondria (UPR mt ) [ 8 , 9 ]. It depends on AAA+ ATPases such as ClpX for activation, substrate selection, and disaggregation, thus converting into a protease [ 10 , 11 , 12 ]. Two heptameric ClpP rings assemble with hexameric ClpX rings on either side into a barrel-shaped unfolding and degradation machine [ 13 ].…”

Proteomic Profiling of Mitochondrial-Derived Vesicles in Brain Reveals Enrichment of Respiratory Complex Sub-assemblies and Small TIM Chaperones

“…Recent studies have also shown that some core subunits (NDUFS1, NDUFV1 and NDUFV2) of the matrix-facing N module of complex I are selectively turned over by the ClpXP protease in the mitochondrial matrix [33][34] . In light of this, it will be important to reconcile how quality control proteases crosstalk with MDV formation and cargo incorporation, particularly since Clpp was identified as enriched in MDVs in our study.…”

Mitochondrial matrix proteases: quality control and beyond

“…Hence, the direct role of ClpXP and LON in metabolic regulation is highly feasible yet understudied. Indeed, the proteomic analysis of ClpXP-deficient models and substrate screens reveals substantial changes in intramitochondrial metabolism and highlight several plausible substrates [103,106,110,112,116].…”