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Mitochondria import nearly all of their approximately 1,000–2,000 constituent proteins from the cytosol across their double-membrane envelope 1 – 5 . Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel 6 – 11 . However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17–Tim23–Tim44) from Saccharomyces cerevisiae . Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis. The cryo-electron microscopy structure of the mitochondrial TIM23 complex from Saccharomyces cerevisiae shows that Tim17 forms a protein translocation path.

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications. HaloTag variants offer distinct brightness and fluorescence lifetimes compared with HaloTag7 when labeled with rhodamines. These variants were used for multiplexed imaging with a single fluorophore and to create lifetime-based cell cycle indicators.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins 1 – 4 . Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex 5 – 9 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 1 – 3 . 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. The high-resolution cryo-electron microscopy structure of the yeast translocase of the outer mitochondrial membrane reveals key features of mitochondrial protein import that are conserved in all eukaryotes.

Sustained mitochondrial fitness relies on coordinated biogenesis and clearance. Both processes are regulated by constant targeting of proteins into the organelle. Thus, mitochondrial protein import sets the pace for mitochondrial abundance and function. However, our understanding of mitochondrial protein translocation as a regulator of longevity remains enigmatic. Here, we targeted the main protein import translocases and assessed their contribution to mitochondrial abundance and organismal physiology. We find that reduction in cellular mitochondrial load through mitochondrial protein import system suppression, referred to as MitoMISS, elicits a distinct longevity paradigm. We show that MitoMISS triggers the mitochondrial unfolded protein response, orchestrating an adaptive reprogramming of metabolism. Glycolysis and de novo serine biosynthesis are causatively linked to longevity, whilst mitochondrial chaperone induction is dispensable for lifespan extension. Our findings extent the pro-longevity role of UPR mt and provide insight, relevant to the metabolic alterations that promote or undermine survival and longevity. Mitochondrial function is linked to lifespan. Here the authors show that inhibition of mitochondrial protein import leads to a reduction in mitochondrial abundance and extends lifespan in Caenorhabditis elegans via activation of glycolysis and de novo serine biosynthesis.

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria after synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. We have determined high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry: pore-forming β-barrel protein Tom40 and four auxiliary α-helical transmembrane proteins. The pore of each Tom40 has an overall negatively charged inner surface attributed to multiple functionally important acidic patches. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Our study reveals the detailed molecular organization of the TOM complex and provides new insights about the mechanism of protein translocation into mitochondria.

Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei. The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

Key messageWe report that discriminate interaction between the expanded mitochondrial chaperone network and variability in their expression might determine their functional specificities and impart robustness to mitochondrial import processes in plants.Mitochondrial Hsp70 (mtHsp70), the central component of the pre-sequence associated motor (PAM) complex, is crucial for the import of proteins to the mitochondrial matrix. Activity of mtHsp70 is regulated by a heterodimeric complex of two J-domain proteins (JDPs), Pam18 and Pam16. Compared to other eukaryotes, plants harbor multiple copies of these JDPs, which posit that plants have an increasingly complex mtHsp70: JDP network in their mitochondrial matrix. Here, we show that although highly similar in sequence, some of the plant JDPs are functionally different. Protein: protein interaction studies including yeast two-hybrid and Bimolecular Fluorescence Complementation revealed that while all the AtPam18s interacted with AtPam16s, the strengths of these promiscuous interactions are variable. Further, down-regulation of AtPAM16L affected seed germination, even in the presence of its seemingly identical paralog, AtPAM16. Knockdown of AtPAM16L caused reduction in mitochondrial number and deregulation of several mitochondrial genes, suggesting towards a specific role of AtPam16L in maintaining mitochondrial homeostasis, especially under stress conditions. Our findings suggest that variations in the spatio-temporal expression, accompanied by discriminate interactions between the JDPs, might be defining the functional specificity of the mtHsp70 co-chaperone machinery and providing resilience to mitochondrial import processes in plants, especially under stress conditions.

Nuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein cytochrome c oxidase assembly factor 7 (COA7), or RESpiratory chain Assembly 1 (RESA1) factor that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency. We found that COA7 requires the mitochondrial IMS import and assembly (MIA) pathway for efficient accumulation in the IMS. We also found that pathogenic mutant versions of COA7 are imported slower than the wild‐type protein, and mislocalized proteins are degraded in the cytosol by the proteasome. Interestingly, proteasome inhibition rescued both the mitochondrial localization of COA7 and complex IV activity in patient‐derived fibroblasts. We propose proteasome inhibition as a novel therapeutic approach for a broad range of mitochondrial pathologies associated with the decreased levels of mitochondrial proteins. Synopsis Biogenesis of the mitochondrial protein COA7 reveals a novel approach for treating diseases associated with the decreased levels of mitochondrial proteins. Proteasome Inhibition of excessive cytosolic degradation of mitochondrial precursors has the potential to restore mitochondrial function. COA7 is synthesized in the cytosol and imported to the intermembrane space of mitochondria by the MIA pathway. COA7‐Y137C and COA7‐exon2Δ protein variants associated with mitochondrial encephalopathy are severely decreased in patient‐derived fibroblasts. Inefficient import of pathogenic COA7 proteins into mitochondria exposes them to an excessive degradation by the proteasome resulting in the impaired mitochondrial respiratory chain assembly. Proteasomal inhibition increases mitochondrial levels of COA7 and restores the function of the respiratory chain. Graphical Abstract Biogenesis of the mitochondrial protein COA7 reveals a novel approach for treating diseases associated with the decreased levels of mitochondrial proteins. Proteasome Inhibition of excessive cytosolic degradation of mitochondrial precursors has the potential to restore mitochondrial function.