Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.

Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging

Mitochondria are essential for the viability of eukaryotic cells as they perform crucial functions in bioenergetics, metabolism and signalling and have been associated with numerous diseases. Recent functional and proteomic studies have revealed the remarkable complexity of mitochondrial protein organization. Protein machineries with diverse functions such as protein translocation, respiration, metabolite transport, protein quality control and the control of membrane architecture interact with each other in dynamic networks. In this Review, we discuss the emerging role of the mitochondrial protein import machinery as a key organizer of these mitochondrial protein networks. The preprotein translocases that reside on the mitochondrial membranes not only function during organelle biogenesis to deliver newly synthesized proteins to their final mitochondrial destination but also cooperate with numerous other mitochondrial protein complexes that perform a wide range of functions. Moreover, these protein networks form membrane contact sites, for example, with the endoplasmic reticulum, that are key for integration of mitochondria with cellular function, and defects in protein import can lead to diseases.

Mitochondrial proteins: from biogenesis to functional networks

Mitochondria contain about 1,000-1,500 proteins that fulfil multiple functions. Mitochondrial proteins originate from two genomes: mitochondrial and nuclear. Hence, proper mitochondrial function requires synchronization of gene expression in the nucleus and in mitochondria and necessitates efficient import of mitochondrial proteins into the organelle from the cytosol. Furthermore, the mitochondrial proteome displays high plasticity to allow the adaptation of mitochondrial function to cellular requirements. Maintenance of this complex and adaptable mitochondrial proteome is challenging, but is of crucial importance to cell function. Defects in mitochondrial proteostasis lead to proteotoxic insults and eventually cell death. Different quality control systems monitor the mitochondrial proteome. The cytosolic ubiquitin-proteasome system controls protein transport across the mitochondrial outer membrane and removes damaged or mislocalized proteins. Concomitantly, a number of mitochondrial chaperones and proteases govern protein folding and degrade damaged proteins inside mitochondria. The quality control factors also regulate processing and turnover of native proteins to control protein import, mitochondrial metabolism, signalling cascades, mitochondrial dynamics and lipid biogenesis, further ensuring proper function of mitochondria. Thus, mitochondrial protein quality control mechanisms are of pivotal importance to integrate mitochondria into the cellular environment.

Quality control of the mitochondrial proteome.

Mitochondria play a pivotal role in bioenergetics and respiratory functions, which are essential for the numerous biochemical processes underpinning cell viability. Mitochondrial morphology changes rapidly in response to external insults and changes in metabolic status via fission and fusion processes (so-called mitochondrial dynamics) that maintain mitochondrial quality and homeostasis. Damaged mitochondria are removed by a process known as mitophagy, which involves their degradation by a specific autophagosomal pathway. Over the last few years, remarkable efforts have been made to investigate the impact on the pathogenesis of Alzheimer's disease (AD) of various forms of mitochondrial dysfunction, such as excessive reactive oxygen species (ROS) production, mitochondrial Ca2+ dyshomeostasis, loss of ATP, and defects in mitochondrial dynamics and transport, and mitophagy. Recent research suggests that restoration of mitochondrial function by physical exercise, an antioxidant diet, or therapeutic approaches can delay the onset and slow the progression of AD. In this review, we focus on recent progress that highlights the crucial role of alterations in mitochondrial function and oxidative stress in the pathogenesis of AD, emphasizing a framework of existing and potential therapeutic approaches.

/pdf/mitochondrial-dysfunction-and-oxidative-stress-in-alzheimer-1mnxemp5h3.pdf

Mitochondrial Dysfunction and Oxidative Stress in Alzheimer's Disease.

Mitochondria are essential organelles of eukaryotic cells. They consist of hundreds of different proteins that exhibit crucial activities in respiration, catabolic metabolism and the synthesis of amino acids, lipids, heme and iron-sulfur clusters. With the exception of a handful of hydrophobic mitochondrially encoded membrane proteins, all these proteins are synthesized on cytosolic ribosomes, targeted to receptors on the mitochondrial surface, and transported across or inserted into the outer and inner mitochondrial membrane before they are folded and assembled into their final native structure. This review article provides a comprehensive overview of the mechanisms and components of the mitochondrial protein import systems with a particular focus on recent developments in the field.

Transport of Proteins into Mitochondria

The majority of organellar proteins are translated on cytosolic ribosomes and must be sorted correctly to function. Targeting routes have been identified for organelles such as peroxisomes and the endoplasmic reticulum (ER). However, little is known about the initial steps of targeting of mitochondrial proteins. In this study, we used a genome-wide screen in yeast and identified factors critical for the intracellular sorting of the mitochondrial inner membrane protein Oxa1. The screen uncovered an unexpected path, termed ER-SURF, for targeting of mitochondrial membrane proteins. This pathway retrieves mitochondrial proteins from the ER surface and reroutes them to mitochondria with the aid of the ER-localized chaperone Djp1. Hence, cells use the expanse of the ER surfaces as a fail-safe to maximize productive mitochondrial protein targeting.

https://science.sciencemag.org/content/sci/361/6407/1118.full.pdf

An ER surface retrieval pathway safeguards the import of mitochondrial membrane proteins in yeast

Oxidative phosphorylation (OXPHOS) complexes consist of nuclear and mitochondrial DNA-encoded subunits. Their biogenesis requires cross-compartment gene regulation to mitigate the accumulation of disproportionate subunits. To determine how human cells coordinate mitochondrial and nuclear gene expression processes, we tailored ribosome profiling for the unique features of the human mitoribosome.We resolve features of mitochondrial translation initiation and identify a small ORF in the 3' UTR of MT-ND5. Analysis of ribosome footprints in five cell types reveals that average mitochondrial synthesis levels correspond precisely to cytosolic levels across OXPHOS complexes, and these average rates reflect the relative abundances of the complexes. Balanced mitochondrial and cytosolic synthesis does not rely on rapid feedback between the two translation systems, and imbalance caused by mitochondrial translation deficiency is associated with the induction of proteotoxicity pathways.Based on our findings, we propose that human OXPHOS complexes are synthesized proportionally to each other, with mitonuclear balance relying on the regulation of OXPHOS subunit translation across cellular compartments, which may represent a proteostasis vulnerability. 

/pdf/balanced-mitochondrial-and-cytosolic-translatomes-underlie-2ic780w5.pdf

Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes

Mitochondrial ribosomes of baker’s yeast contain at least 78 protein subunits. All but one of these proteins are nuclear-encoded, synthesized on cytosolic ribosomes, and imported into the matrix fo...

Import of ribosomal proteins into yeast mitochondria.

Mitochondrial biogenesis relies on the synthesis of hundreds of different precursor proteins in the cytosol and their subsequent import into the organelle. Recent studies suggest that the surface of the endoplasmic reticulum (ER) actively contributes to the targeting of some mitochondrial precursors. In the past, in vitro import experiments with isolated mitochondria proved to be extremely powerful to elucidate the individual reactions of the mitochondrial import machinery. However, this in vitro approach is not well suited to study the influence of non-mitochondrial membranes. In this study, we describe an in vitro system using semi-intact yeast cells to test a potential import relevance of the ER proteins Erg3, Lcb5 and Ssh1, all being required for efficient mitochondrial respiration. We optimized the conditions of this experimental test system and found that cells lacking Ssh1, a paralog of the Sec61 translocation pore, show a reduced import efficiency of mitochondrial precursor proteins. Our results suggest that Ssh1, directly or indirectly, increases the efficiency of the biogenesis of mitochondrial proteins. Our findings are compatible with a functional interdependence of the mitochondrial and the ER protein translocation systems.

Katja G. Hansen

Papers

An ER surface retrieval pathway safeguards the import of mitochondrial membrane proteins in yeast

Transport of Proteins into Mitochondria

Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes

Import of ribosomal proteins into yeast mitochondria.

In vitro import experiments with semi-intact cells suggest a role of the Sec61 paralog Ssh1 in mitochondrial biogenesis.