Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions.

Aurelio Reyes,Alan J Robinson,Laura Melchionda,Massimo Zeviani,Alberto Burlina,Daniele Ghezzi

doi:10.15252/emmm.201708698

Abstract

TIMM50 is an essential component of the TIM23 complex, the mitochondrial inner membrane machinery that imports cytosolic proteins containing a mitochondrial targeting presequence into the mitochondrial inner compartment. Whole exome sequencing (WES) identified compound heterozygous pathogenic mutations in TIMM50 in an infant patient with rapidly progressive, severe encephalopathy. Patient fibroblasts presented low levels of TIMM50 and other components of the TIM23 complex, lower mitochondrial membrane potential, and impaired TIM23‐dependent protein import. As a consequence, steady‐state levels of several components of mitochondrial respiratory chain were decreased, resulting in decreased respiration and increased ROS production. Growth of patient fibroblasts in galactose shifted energy production metabolism toward oxidative phosphorylation (OxPhos), producing an apparent improvement in most of the above features but also increased apoptosis. Complementation of patient fibroblasts with TIMM50 improved or restored these features to control levels. Moreover, RNASEH1 and ISCU mutant fibroblasts only shared a few of these features with TIMM50 mutant fibroblasts. Our results indicate that mutations in TIMM50 cause multiple mitochondrial bioenergetic dysfunction and that functional TIMM50 is essential for cell survival in OxPhos‐dependent conditions.

Highlights

The mammalian mitochondrial proteome is estimated to contain about 1,500 proteins (Meisinger et al, 2008), but only 13 of them are encoded by the mitochondrial DNA and synthesized in the mitochondrial matrix
We observed no significant increase in apoptosis in glucose; there was a fivefold increase in apoptotic cells when patient fibroblasts were grown in galactose medium while no such change was detected in controls; importantly, this phenotype was lost after transduction with TIMM50 (Fig 6C and D)
Analysis of oxidative phosphorylation (OxPhos) constituents of all five complexes revealed that significant changes compared to control were only detected for SDHB and COX4I1 in ISCU mutant fibroblasts grown in glucose and MT-CO1 in both RNASEH1 and ISCU mutant fibroblasts grown in galactose (Fig 7C and Appendix Fig S9)

Summary

Introduction

The mammalian mitochondrial proteome is estimated to contain about 1,500 proteins (Meisinger et al, 2008), but only 13 of them are encoded by the mitochondrial DNA (mtDNA) and synthesized in the mitochondrial matrix. The remaining proteins are encoded by nuclear genes, synthesized on cytosolic ribosomes and imported into mitochondria by specific protein import machineries located on both the outer and the inner mitochondrial membranes (OMM and IMM, respectively; Sokol et al, 2014). The translocase of the outer mitochondrial membrane (TOM) complex is the common entry gate for all mitochondrial precursor proteins and, in humans, it consists of eight subunits (Fig 1A). The channelforming subunit is TOMM40 while TOMM22 both stabilizes the complex and interacts with subunits of the translocase of the inner mitochondrial membrane (TIM) complex. Additional small subunits, TOMM5, TOMM6, and TOMM7, are involved in the assembly and stability of the TOM complex (Kato & Mihara, 2008)

Methods

Results

Conclusion