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.
Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol ...and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics.
The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins
. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex
at 3.8-Å ...resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes
. Each Tom40 β-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.
Mitochondria perform central functions in cellular bioenergetics, metabolism, and signaling, and their dysfunction has been linked to numerous diseases. The available studies cover only part of the ...mitochondrial proteome, and a separation of core mitochondrial proteins from associated fractions has not been achieved. We developed an integrative experimental approach to define the proteome of yeast mitochondria. We classified > 3,300 proteins of mitochondria and mitochondria-associated fractions and defined 901 high-confidence mitochondrial proteins, expanding the set of mitochondrial proteins by 82. Our analysis includes protein abundance under fermentable and nonfermentable growth, submitochondrial localization, single-protein experiments, and subcellular classification of mitochondria-associated fractions. We identified mitochondrial interactors of respiratory chain supercomplexes, ATP synthase, AAA proteases, the mitochondrial contact site and cristae organizing system (MICOS), and the coenzyme Q biosynthesis cluster, as well as mitochondrial proteins with dual cellular localization. The integrative proteome provides a high-confidence source for the characterization of physiological and pathophysiological functions of mitochondria and their integration into the cellular environment.
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•Classification of > 3,300 proteins of mitochondria and associated fractions•High-confidence mitochondrial proteome with absolute quantification and topology•Interactors of oxidative phosphorylation complexes and cristae organizing system•Identification of system linking respiratory chain and AAA quality control
Morgenstern et al. describe an integrative organelle proteomics analysis to define the mitochondrial proteome in baker’s yeast. The study provides a quantitative footprint of the proteome and its dynamics under different conditions. The results expand the set of proteins assigned to the mitochondria and provide a resource for future mitochondrial research.
Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human ...mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.
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•Human mitochondrial high-confidence proteome with >1,100 proteins (MitoCoP)•Mitochondria-specific protein copy numbers and half-lives•Interactors of protein translocases and oxidative phosphorylation assembly factors•>40% of mitochondrial proteome linked to human diseases
Mitochondria are crucial for cellular energy metabolism and human health. Morgenstern et al. present a high-confidence protein compendium of human mitochondria including mitochondria-specific protein copy numbers and half-lives. They identify interactors of key mitochondrial protein machineries and link >40% of the mitochondrial proteome to human diseases.
The biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria requires the insertion of β-barrel proteins into the outer membranes. Homologous Omp85 proteins are essential for membrane ...insertion of β-barrel precursors. It is unknown if precursors are threaded through the Omp85-channel interior and exit laterally or if they are translocated into the membrane at the Omp85-lipid interface. We have mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by β-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase may represent a basic mechanism for membrane insertion of β-barrel proteins.
Communication between organelles is crucial for eukaryotic cells to function as one coherent unit. An important means of communication is through membrane contact sites, where two organelles come ...into close proximity allowing the transport of lipids and small solutes between them. Contact sites are dynamic in size and can change in response to environmental or cellular stimuli; however, how this is regulated has been unclear. Here, we show that Saccharomyces cerevisiae Lam6 resides in several central contact sites: ERMES (ER/mitochondria encounter structure), vCLAMP (vacuole and mitochondria patch), and NVJ (nuclear vacuolar junction). We show that Lam6 is sufficient for expansion of contact sites under physiological conditions and necessary for coordination of contact site size. Given that Lam6 is part of a large protein family and is conserved in vertebrates, our work opens avenues for investigating the underlying principles of organelle communication.
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•Lam6 is localized to three major cellular contacts: ERMES, vCLAMP, and NVJ•Lam6 is a GRAM domain protein conserved from yeast to humans•Overexpression of Lam6 results in the expansion of all three contact sites•Lam6 is essential for the cross-talk between ERMES and vCLAMP
Elbaz-Alon et al. find that yeast Lam6, which is part of a conserved protein family, is found in three types of organellar contact sites, ERMES, vCLAMP, and NVJ. Overexpression of Lam6 is sufficient to expand these contact sites and its expression is necessary for cross-regulation of contact site size.
Significance Mitochondria contain several hundreds of proteins. The mitochondrial content is regulated by the uptake and degradation of proteins. Stabilization of protein structure by disulfide bonds ...was proposed to drive protein accumulation in the intermembrane space of mitochondria. However, it remained unknown if structural alterations could lead to protein escape through the physiological barrier formed by the outer mitochondrial membrane. In this work, we present evidence for size-dependent retrograde movement of mitochondrial proteins to the cytosol. We identify the translocase of the outer mitochondrial membrane channel protein Tom40 as an exit route. Our results indicate that the retro-translocation serves as an important surveillance mechanism that regulates the abundance of intermembrane space proteins in response to changes in cellular physiology.
The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.
Mitochondria fulfill central functions in cellular energetics, metabolism, and signaling. The outer membrane translocator complex (the TOM complex) imports most mitochondrial proteins, but its ...architecture is unknown. Using a cross-linking approach, we mapped the active translocator down to single amino acid residues, revealing different transport paths for preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel to recruit chaperones from the intermembrane space that guide the transfer of hydrophobic preproteins. The translocator contains three Tom40 β-barrel channels sandwiched between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex exchanges with a dimeric isoform to assemble new TOM complexes. Dynamic coupling of α-helical receptors, β-barrel chennels, and chaperones generates a versatile machinery that transports about 1000 different proteins.
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