Global Proteome of LonP1+/- Mouse Embryonal Fibroblasts Reveals Impact on Respiratory Chain, but No Interdependence between Eral1 and Mitoribosomes.

Jana Key,Juliana Heidler,Georg Auburger,Ilka Wittig,Clea Bárcena,Aneesha Kohli,Carlos López-Otín

doi:10.3390/ijms20184523

Abstract

Research on healthy aging shows that lifespan reductions are often caused by mitochondrial dysfunction. Thus, it is very interesting that the deletion of mitochondrial matrix peptidase LonP1 was observed to abolish embryogenesis, while deletion of the mitochondrial matrix peptidase Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (ClpP) prolonged survival. To unveil the targets of each enzyme, we documented the global proteome of LonP1+/− mouse embryonal fibroblasts (MEF), for comparison with ClpP−/− depletion. Proteomic profiles of LonP1+/− MEF generated by label-free mass spectrometry were further processed with the STRING (Search tool for the retrieval of interacting genes) webserver Heidelberg for protein interactions. ClpP was previously reported to degrade Eral1 as a chaperone involved in mitoribosome assembly, so ClpP deficiency triggers the accumulation of mitoribosomal subunits and inefficient translation. LonP1+/− MEF also showed Eral1 accumulation, but no systematic effect on mitoribosomal subunits. In contrast to ClpP−/− profiles, several components of the respiratory complex-I membrane arm, of the glutathione pathway and of lysosomes were accumulated, whereas the upregulation of numerous innate immune defense components was similar. Overall, LonP1, as opposed to ClpP, appears to have no effect on translational machinery, instead it shows enhanced respiratory dysfunction; this agrees with reports on the human CODAS syndrome (syndrome with cerebral, ocular, dental, auricular, and skeletal anomalies) caused by LonP1 mutations.

Highlights

Mitochondria are double membraned subcellular organelles which are implicated in multiple cellular processes
Four energy (ATP)-dependent proteolytic systems exist, with Lonp1 (Lon Protease Homolog 1) in the matrix being the simplest example as a soluble homo-hexamer single-ring with each subunit containing an AAA+ (ATPases Associated with diverse cellular Activities) ATPase plus a serine peptidase domain
ClpXP protease in the matrix is soluble but consists of two stacked heptameric ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) serine peptidase rings attached to caps of hexameric ClpX (Caseinolytic Mitochondrial Matrix Peptidase Chaperone Subunit) AAA+ ATPase rings at either end

Summary

Introduction

Mitochondria are double membraned subcellular organelles which are implicated in multiple cellular processes. In addition to their main role in the production of energy through being the organelle housing oxidative phosphorylation and the citric acid cycle, mitochondria exert many other functions These range from cellular calcium homeostasis [1], regulation of reactive oxygen species (ROS), assembly of iron–sulfur-cluster and heme, to participation in innate immune responses of any cell type. While the deficiency of LonP1 on the one hand and the subsequent accumulation of misfolded proteins in the mitochondria trigger an increase of Pink and the engulfment/elimination of mitochondria via the autophago–lysosomal pathway [48], the deficiency of ClpP, on the other hand, triggers selective changes in the cytosolic proteasome [46] In both cases, the mitochondrial dysfunction caused by either LonP1 or ClpP depletion activates the Rig-I-like receptor pathway of the innate immune system, perhaps by altered binding of lactate and hexokinase-2 to the mitochondrial antiviral signaling protein Mavs [46,49,50,51]. The comparison with previously reported ClpP−/− expression profiles may help to identify substrates that are selectively degraded by each of these proteases

Results

Discussion

Cell Culture

Proteomics

Bioinformatic Analyses