Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.

Sven Dennerlein,Sabine Poerschke,Silke Oeljeklaus,Thomas Langer,Cong Wang,Johannes Sattmann,Stefan Jakobs,Elisa Hanitsch,Diana Bauermeister,Ricarda Richter-Dennerlein,Stefan Stoldt,Bettina Warscheid,Peter Rehling

doi:10.7554/elife.68213

Abstract

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

Highlights

Mitochondria play key roles in a plethora of cellular processes such as signaling processes, metabolism, and energy production (Pfanner et al, 2019)
To identify factors that are associated with the mt-ribosome and thereby contribute to oxidative phosphorylation (OXPHOS) biogenesis, we generated a human HEK293-Flp-InTM T-RexTM (HEK293T) cell line that enables inducible expression of a FLAG-tagged version of the ribosomal subunit mL62FLAG (Richter-D ennerlein et al, 2016). mL62 is a component of the 39S large ribosomal subunit (39S mtLSU) (Richter et al, 2010; Brown et al, 2014; Greber et al, 2014; Busch et al, 2019)
As SMIM4 and C12ORF73 were present in early assembly intermediates and their loss led to a reduction of the cytochrome c reductase, we addressed at which stage a loss of C12ORF73 affects cytochrome c reductase assembly

Summary

Introduction

Mitochondria play key roles in a plethora of cellular processes such as signaling processes, metabolism, and energy production (Pfanner et al, 2019) Among this multitude of functions, cellular energy conversation by oxidative phosphorylation (OXPHOS) is a hallmark. The human mitochondrial genome (mtDNA) encodes 2 rRNAs, 22 tRNAs, and 13 proteins These proteins are synthesized by membrane-a ssociated mitochondrial ribosomes (mt-ribosome) to insert their translation products into the membrane co-t ranslationally (Englmeier et al, 2017; Pfeffer et al, 2015). These subunits have to engage with nuclear-encoded, imported subunits to form functional enzyme machineries. This process requires a large number of chaperone-like assembly factors, which promote the maturation of the complexes through a number of assembly intermediates

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