Import Receptor Tom70 Research Articles

DYRK1A signalling synchronizes the mitochondrial import pathways for metabolic rewiring

Mitochondria require an extensive proteome to maintain a variety of metabolic reactions, and changes in cellular demand depend on rapid adaptation of the mitochondrial protein composition. The TOM complex, the organellar entry gate for mitochondrial precursors in the outer membrane, is a target for cytosolic kinases to modulate protein influx. DYRK1A phosphorylation of the carrier import receptor TOM70 at Ser91 enables its efficient docking and thus transfer of precursor proteins to the TOM complex. Here, we probe TOM70 phosphorylation in molecular detail and find that TOM70 is not a CK2 target nor import receptor for MIC19 as previously suggested. Instead, we identify TOM20 as a MIC19 import receptor and show off-target inhibition of the DYRK1A-TOM70 axis with the clinically used CK2 inhibitor CX4945 which activates TOM20-dependent import pathways. Taken together, modulation of DYRK1A signalling adapts the central mitochondrial protein entry gate via synchronization of TOM70- and TOM20-dependent import pathways for metabolic rewiring. Thus, DYRK1A emerges as a cytosolic surveillance kinase to regulate and fine-tune mitochondrial protein biogenesis.

Nascent mitochondrial proteins initiate the localized condensation of cytosolic protein aggregates on the mitochondrial surface

Eukaryotes organize cellular contents into membrane-bound organelles and membrane-less condensates, for example, protein aggregates. An unsolved question is why the ubiquitously distributed proteins throughout the cytosol give rise to spatially localized protein aggregates on the organellar surface, like mitochondria. We report that the mitochondrial import receptor Tom70 is involved in the localized condensation of protein aggregates in budding yeast and human cells. This is because misfolded cytosolic proteins do not autonomously aggregate in vivo; instead, they are recruited to the condensation sites initiated by Tom70's substrates (nascent mitochondrial proteins) on the organellar membrane using multivalent hydrophobic interactions. Knocking out Tom70 partially impairs, while overexpressing Tom70 increases the formation and association between cytosolic protein aggregates and mitochondria. In addition, ectopic targeting Tom70 and its substrates to the vacuole surface is able to redirect the localized aggregation from mitochondria to the vacuolar surface. Although other redundant mechanisms may exist, this nascent mitochondrial proteins-based initiation of protein aggregation likely explains the localized condensation of otherwise ubiquitously distributed molecules on the mitochondria. Disrupting the mitochondrial association of aggregates impairs their asymmetric retention during mitosis and reduces the mitochondrial import of misfolded proteins, suggesting a proteostasis role of the organelle-condensate interactions.

The role of the individual TOM subunits in the association of PINK1 with depolarized mitochondria

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinson’s disease. PINK1, which is mutated in some cases of familial Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria.Key messagesTOM70 is the main receptor for the import of PINK1 into mitochondria.TOM20 plays only a minor role in PINK1 recognition at the organellar outer membrane.PINK1 association with the TOM complex is reduced upon knock-down of TOM7.The lumen of the TOM pore is crucial for PINK1 association with the outer membrane. TcPINK1 blocks the TOM pore in depolarized mitochondria.

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.

Synthetic Mitochondria-Targeting Peptides Incorporating α-Aminoisobutyric Acid with a Stable Amphiphilic Helix Conformation in Plant Cells.

In the genetic modification of plant cells, the mitochondrion is an important target in addition to the nucleus and plastid. However, gene delivery into the mitochondria of plant cells has yet to be established by conventional methods, such as particle bombardment, because of the small size and high mobility of mitochondria. To develop an efficient mitochondria-targeting signal (MTS) that functions in plant cells, we designed the artificial peptide (LURL)3 and its analogues, which periodically feature hydrophobic α-aminoisobutyric acid (Aib, U) and cationic arginine (R), considering the consensus motif recognized by the mitochondrial import receptor Tom20. Circular dichroism measurements and molecular dynamics simulation studies revealed that (LURL)3 had a propensity to form a stable α-helix in 0.1 M phosphate buffer solution containing 1.0 wt % sodium dodecyl sulfate. After internalization into plant cells via particle bombardment, (LURL)3 revealed highly selective accumulation in the mitochondria, whereas its analogue (LARL)3 was predominantly located in the vacuoles in addition to mitochondria. The high selectivity of (LURL)3 can be attributed to the incorporation of Aib, which promotes the hydrophobic interaction between the MTS and Tom20 by increasing the hydrophobicity and helicity of (LURL)3. The present study provided a prospective mitochondrial targeting system using the simple design of artificial peptides.

Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos.

SummaryIn mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1, 2, 3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4, 5, 6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7, 8, 9, 10, 11, 12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.

Development of GMP-1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease.

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aβ) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria‐destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP‐1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP‐1 treatment of SH‐SY5Y cells results in decrease in mitochondria‐associated APP and protects SH‐SY5Y cells from toxic effect of Aβ1‐42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP‐1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.

Mitochondria‐targeting peptide from Hibiscus sabdariffa

Cysteine‐rich peptides (CRPs) are hyperstable, multi‐disulfide‐constrained peptides. Structurally, CRPs contain evolutionary conserved disulfided scaffolds, often with functional intercysteine loops which contribute to their multiple biological activities. Previously, we have identified Hibiscus sabdariffa as a rich source of CRPs. Hibiscus sabdariffa, commonly known as roselle, is a South‐east Asian plant. It is used as a folk medicine for treating age‐related conditions and diseases. The prototypic CRP derived from roselle is roseltide rT1, a 27‐residue cysteine‐rich peptide with six cysteine forming four intercycteine loops. We showed that Loop 1 contributes to the inhibitory activities against human neutrophil elastase. However the functions of other loops, individually or in combination have not been determined. Here we report that roseltide rT1 is a mitochondria‐targeting peptide with bioenergetic properties. We showed that roseltide rT1 interacts with the mitochondrial import receptor TOM20 for its localization to the mitochondria. We prepared the linear precursor of roseltide rT1 using a stepwise solid‐phase method followed by oxidative folding to its native state. The conformation of synthetic rT1 was confirmed using reversed‐phase high‐pressure liquid chromatography and nuclear magnetic resonance spectroscopy. Using N‐hydroxysuccinimide ester, N‐terminal biotin‐ and Cy3‐labeled roseltide rT1 were generated for biological assays. Our results showed that mitochondrial depolarization induced by mitochondria uncoupler carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone does not abolish the mitochondrial localization of Cy3‐rT1. In silico docking suggested that the leucine‐rich loop 2 of roseltide rT1 interacts with TOM20. Using pull‐down assay, biotin‐rT1 was shown to interact with TOM20. Using live‐cell confocal microscopy, Cy3‐rT1 was shown to co‐localize with TOM20‐GFP in stably‐transfected HEK293 cells. Additionally, the localization of Cy3‐rT1 with MitoTracker Green was reduced in TOM20‐knocked‐down HepG2 cells compared to control. Taken together, this study shows that the mitochondrial import receptor TOM20 plays an important role for the localization of roseltide rT1 to the mitochondria.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐ Synzyme and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The cytosolic cochaperone Sti1 is relevant for mitochondrial biogenesis and morphology.

Most mitochondrial proteins are synthesized in the cytosol prior to their import into the organelle. It is commonly accepted that cytosolic factors are required for delivering precursor proteins to the mitochondrial surface and for keeping newly synthesized proteins in an import-competent conformation. However, the identity of such factors and their defined contribution to the import process are mostly unknown. Using a presequence-containing model protein and a site-directed photo-crosslinking approach in yeast cells we identified the cytosolic chaperones Hsp70 (Ssa1) and Hsp90 (Hsp82) as well as their cochaperones, Sti1 and Ydj1, as putative cytosolic factors involved in mitochondrial protein import. Deletion of STI1 caused both alterations in mitochondrial morphology and lower steady-state levels of a subset of mitochondrial proteins. In addition, double deletion of STI1 with the mitochondrial import factors, MIM1 or TOM20, showed a synthetic growth phenotype indicating a genetic interaction of STI1 with these genes. Moreover, recombinant cytosolic domains of the import receptors Tom20 and Tom70 were able to bind in vitro Sti1 and other cytosolic factors. In summary, our observations point to a, direct or indirect, role of Sti1 for mitochondrial functionality.

Abstract 3530: Hsp90-Tom70 interaction facilitates ATM localization to mitochondria and induces mitochondrial autophagy

Abstract The serine/threonine protein kinase Ataxia telangiectasia mutated (ATM), plays a critical role in DNA damage response specifically to agents that induce DNA double strand breaks. Recently, mitochondrial autophagy (mitophagy)-associated non-nuclear functions have been attributed to ATM in AT fibroblast (Valentin-Vega YA et al., Blood 119: 1490; 2012). Our investigations suggested similar role of ATM in mantle cell lymphoma (MCL) cell lines (Sarkar, A et al., Proc. AACR, 74: Abstract # 313; 2014). This is clinically important, since ATM is lost in >50% of primary malignant B-cells in MCL patients. To identify the role of ATM in mitophagy, using MCL cell lines, we established that a fraction of ATM protein is located within mitochondria. The proportion of mitochondrial ATM increases following mitophagy induced by mitochondrial uncoupler CCCP or FCCP in ATM proficient cell lines. This process is compromised in MCL cells lacking ATM. However how ATM enters mitochondria is not known. Using quantitative liquid chromatography mass spectrometry analysis (LC/MS), we identified new ATM interacting proteins in HEK293 cells that facilitate mitochondrial transport of ATM. These new ATM interacting proteins (Hsp90, Hsp70 and the outer mitochondrial translocation (TOM) complex, including Tom70) are unrelated to DNA damage response. The physical association of ATM with Hsp90 and Tom70 was confirmed by co-immunoprecipitation experiments while ATM-Hsp70 interaction was mostly nonspecific. Additionally, using kinase dead FLAG-ATM construct in HEK293 cells or pharmacologic (KU55933) inhibition of ATM kinase activity in ATM proficient Jeko-1 and Mino MCL cell lines, we demonstrate that ATM phosphorylation is dispensable to enforce mitophagic exclusion of nonfunctional mitochondria since non-phosphorylated ATM can still bound to Hsp90 and induce mitophagy. These MCL cell lines when treated with an Hsp90 inhibitor; 17-AAG either alone or in combination with CCCP; abolished mitochondrial ATM localization and are defective in CCCP-induced mitochondrial clearance suggesting extra-mitochondrial ATM enters mitochondria via Hsp90 interaction. Further analysis revealed ATM-Hsp90 loosely associate with outer mitochondrial import receptor Tom70 via Hsp90 interaction and this targeting preference between Hsp90-Tom70 is abolished following deletion of the cytosolic tricopeptide repeat (TPR) clamp domain of Tom70 entailing that ATM-Hsp90-Tom70 complex may be required for ATM entry inside mitochondria. Moreover, trypsin digested purified mitochondrial fraction from HeLa cells revealed the entry of C terminal ATM protein within mitochondria while N terminal part remained in the cytosolic fraction. The molecular model of ATM's role in mitophagy and its biological consequence in ATM deficient MCL will be discussed. Citation Format: Aloke K. Sarkar, Tej K. Pandita, Varsha Gandhi. Hsp90-Tom70 interaction facilitates ATM localization to mitochondria and induces mitochondrial autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3530.

Characterization of the targeting signal in mitochondrial β-barrel proteins.

Mitochondrial β-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized β-hairpin motif is this long searched for signal. We demonstrate that a synthetic β-hairpin peptide competes with the import of mitochondrial β-barrel proteins and that proteins harbouring a β-hairpin peptide fused to passenger domains are targeted to mitochondria. Furthermore, a β-hairpin motif from mitochondrial proteins targets chloroplast β-barrel proteins to mitochondria. The mitochondrial targeting depends on the hydrophobicity of the β-hairpin motif. Finally, this motif interacts with the mitochondrial import receptor Tom20. Collectively, we reveal that β-barrel proteins are targeted to mitochondria by a dedicated β-hairpin element, and this motif is recognized at the organelle surface by the outer membrane translocase.

α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson's disease.

α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson's disease (PD), and the two appear to be related. Mitochondrial dysfunction leads to accumulation and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment, but the basis for this bidirectional interaction remains obscure. We now report that certain posttranslationally modified species of α-synuclein bind with high affinity to the TOM20 (translocase of the outer membrane 20) presequence receptor of the mitochondrial protein import machinery. This binding prevented the interaction of TOM20 with its co-receptor, TOM22, and impaired mitochondrial protein import. Consequently, there were deficient mitochondrial respiration, enhanced production of reactive oxygen species, and loss of mitochondrial membrane potential. Examination of postmortem brain tissue from PD patients revealed an aberrant α-synuclein-TOM20 interaction in nigrostriatal dopaminergic neurons that was associated with loss of imported mitochondrial proteins, thereby confirming this pathogenic process in the human disease. Modest knockdown of endogenous α-synuclein was sufficient to maintain mitochondrial protein import in an in vivo model of PD. Furthermore, in in vitro systems, overexpression of TOM20 or a mitochondrial targeting signal peptide had beneficial effects and preserved mitochondrial protein import. This study characterizes a pathogenic mechanism in PD, identifies toxic species of wild-type α-synuclein, and reveals potential new therapeutic strategies for neuroprotection.

Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER-mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES-related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane.

Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins.

A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1Δ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins.

Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure

Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal (SSM) mitochondria harvested from rats who have received 4weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biologic system. SSM mitochondria were harvested, and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with immobilized metal ion affinity chromatography. The phosphopeptides were analyzed by ion trap liquid chromatography-tandem mass spectrometry, and the phosphoproteins identified via database searches. Based on MS/MS and MS(3) data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example, long-chain specific acyl-CoA dehydrogenase, Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling toward an integrated understanding of the role of mitochondrial phosphorylation in heart failure.

The ubiquitin-conjugating enzymes UBE2N, UBE2L3 and UBE2D2/3 are essential for Parkin-dependent mitophagy.

Depolarized mitochondria are degraded by mitophagy in a process that depends on the Parkinson's disease gene products PINK1 and Parkin. This is accompanied by ubiquitylation of several mitochondrial substrates. The roles of E2 ubiquitin-conjugating enzymes (UBE2) in mitophagy are poorly understood. Here, we investigate a set of UBE2 enzymes that might regulate Parkin-mediated mitophagy. Knockdown of the E2 enzymes UBE2N, UBE2L3 or UBE2D2 and UBE2D3 (UBE2D2/3) significantly reduced autophagic clearance of depolarized mitochondria. However, this did not interfere with mitochondrial PINK1 stabilization and Parkin translocation. UBE2N knockdown prevented specifically K63-linked ubiquitylation at mitochondrial sites. Nevertheless, polyubiquitin and p62 (officially known as SQSTM1) were still found on mitochondria after individual UBE2 knockdown. Knockdown of all of these UBE2s together significantly reduced mitochondrial polyubiquitylation and p62 recruitment. Moreover, reduced ubiquitylation of mitofusins, the mitochondrial import receptor subunits TOM20 and TOM70, the voltage-dependent anion channel protein 1 and Parkin was observed in cells silenced for all of these UBE2s. A version of Parkin with a mutation in the active site (C431S) failed to ubiquitylate these mitochondrial substrates even in the presence of UBE2s. We conclude that UBE2N, UBE2L3 and UBE2D2/3 synergistically contribute to Parkin-mediated mitophagy.

The Role of Djp1 in Import of the Mitochondrial Protein Mim1 Demonstrates Specificity between a Cochaperone and Its Substrate Protein

A special group of mitochondrial outer membrane proteins spans the membrane once, exposing soluble domains to both sides of the membrane. These proteins are synthesized in the cytosol and then inserted into the membrane by an unknown mechanism. To identify proteins that are involved in the biogenesis of the single-span model protein Mim1, we performed a high-throughput screen in yeast. Two interesting candidates were the cytosolic cochaperone Djp1 and the mitochondrial import receptor Tom70. Our results indeed demonstrate a direct interaction of newly synthesized Mim1 molecules with Tom70. We further observed lower steady-state levels of Mim1 in mitochondria from djp1Δ and tom70 tom71Δ cells and massive mislocalization of overexpressed GFP-Mim1 to the endoplasmic reticulum in the absence of Djp1. Importantly, these phenotypes were observed specifically for the deletion of DJP1 and were not detected in mutant cells lacking any of the other cytosolic cochaperones of the Hsp40 family. Furthermore, the djp1Δ tom70Δ tom71Δ triple deletion resulted in a severe synthetic sick/lethal growth phenotype. Taking our results together, we identified Tom70 and Djp1 as crucial players in the biogenesis of Mim1. Moreover, the involvement of Djp1 provides a unique case of specificity between a cochaperone and its substrate protein.

Tom70 is essential for PINK1 import into mitochondria.

PTEN induced kinase 1 (PINK1) is a serine/threonine kinase in the outer membrane of mitochondria (OMM), and known as a responsible gene of Parkinson's disease (PD). The precursor of PINK1 is synthesized in the cytosol and then imported into the mitochondria via the translocase of the OMM (TOM) complex. However, a large part of PINK1 import mechanism remains unclear. In this study, we examined using cell-free system the mechanism by which PINK1 is targeted to and assembled into mitochondria. Surprisingly, the main component of the import channel, Tom40 was not necessary for PINK1 import. Furthermore, we revealed that the import receptor Tom70 is essential for PINK1 import. In addition, we observed that although PINK1 has predicted mitochondrial targeting signal, it was not processed by the mitochondrial processing peptidase. Thus, our results suggest that PINK1 is imported into mitochondria by a unique pathway that is independent of the TOM core complex but crucially depends on the import receptor Tom70.

Identification of genes required for alternative oxidase production in the Neurospora crassa gene knockout library.

The alternative oxidase (AOX) of Neurospora crassa transfers electrons from ubiquinol to oxygen. The enzyme is not expressed under normal conditions. However, when the function of the standard electron transport chain is compromised, AOX is induced, providing cells with a means to continue respiration and growth. Induction of the enzyme represents a form of retrograde regulation because AOX is encoded by a nuclear gene that responds to signals produced from inefficiently functioning mitochondria. To identify genes required for AOX expression, we have screened the N. crassa gene knockout library for strains that are unable to grow in the presence of antimycin A, an inhibitor of complex III of the standard electron transport chain. From the 7800 strains containing knockouts of different genes, we identified 62 strains that have reduced levels of AOX when grown under conditions known to induce the enzyme. Some strains have virtually no AOX, whereas others have only a slight reduction of the protein. A broad range of seemingly unrelated functions are represented in the knockouts. For example, we identified transcription factors, kinases, the mitochondrial import receptor Tom70, three subunits of the COP9 signalosome, a monothiol glutaredoxin, and several hypothetical proteins as being required for wild-type levels of AOX production. Our results suggest that defects in many signaling or metabolic pathways have a negative effect on AOX expression and imply that complex systems control production of the enzyme.