High-resolution imaging reveals compartmentalization of mitochondrial protein synthesis in cultured human cells

Matthew Zorkau,Christin A Albus,Rolando Berlinguer-Palmini,Zofia M A Chrzanowska-Lightowlers,Robert N Lightowlers

doi:10.1073/pnas.2008778118

Matthew Zorkau, Christin A Albus + Show 3 more

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https://doi.org/10.1073/pnas.2008778118

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Abstract

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multisubunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially encoded components are also integral members of these complexes, where does protein synthesis occur? As transcription, mRNA processing, maturation, and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites? We have adapted a click chemistry-based method coupled with stimulated emission depletion nanoscopy to address these questions. We report that, in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis is detected at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.

Highlights

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix
It comprises 1) the inner boundary membrane (IBM) that is closely apposed to the outer mitochondrial membrane (OMM) and where contact sites containing the machinery for importing proteins from the cytosol are found [6, 7]; 2) highly invaginated cristae membranes (CMs) that house the majority of the oxidative phosphorylation (OXPHOS) complexes [8,9,10], in particular the FoF1 ATP synthase, dimers of which help generate the architecture of the membranes [11]; and 3) large (10 to 40 nm) complexes termed cristae junctions (CJs) composed of the Mitochondrial contact site and Cristae Organizing System (MICOS) [12,13,14], which forms the cristae and separates them from the IBM
It is tempting to infer that a subset of HPGylated mt-tRNAMet could have been formylated by mitochondrial methionyl-tRNA formyltransferase, mimicking the endogenous initiation mechanism [33], but recent data suggest that, under certain circumstances, initiation in mitochondria may occur without formylation of an initiating mt-tRNAMet [34]

Summary

Introduction

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. It is clear that the inner mitochondrial membrane (IMM) has highly defined regions It comprises 1) the inner boundary membrane (IBM) that is closely apposed to the outer mitochondrial membrane (OMM) and where contact sites containing the machinery for importing proteins from the cytosol are found [6, 7]; 2) highly invaginated cristae membranes (CMs) that house the majority of the OXPHOS complexes [8,9,10], in particular the FoF1 ATP synthase, dimers of which help generate the architecture of the membranes [11]; and 3) large (10 to 40 nm) complexes termed cristae junctions (CJs) composed of the Mitochondrial contact site and Cristae Organizing System (MICOS) [12,13,14], which forms the cristae and separates them from the IBM. We report: 1) a method for visualizing protein synthesis in human mitochondria at super resolution; 2) that synthesis is enriched at cristae membranes, in preference to the IBM; and 3) that sites of translation are spatially separated from RNA granules where RNA processing, maturation, and mitoribosomal assembly occur As the majority of OXPHOS components are imported and need to be integrated in assembly with the mtDNA-encoded components, where does intramitochondrial translation occur? Here we report: 1) a method for visualizing protein synthesis in human mitochondria at super resolution; 2) that synthesis is enriched at cristae membranes, in preference to the IBM; and 3) that sites of translation are spatially separated from RNA granules where RNA processing, maturation, and mitoribosomal assembly occur

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