Podcasts about tim23

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.26.530105v1?rss=1 Authors: Bi, P. Y., Killackey, S. A., Schweizer, L., Arnoult, D., Philpott, D. J., Girardin, S. E. Abstract: Mitochondrial stress inducers, such as the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the ATPase pump inhibitor oligomycin, trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged forms of DELE1 showed that these stresses induced the accumulation of a cleaved form of DELE1, DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non-depolarizing MPIS. We further showed that DELE1 specifically senses MPIS triggered by the inhibition of the TIM23 complex at the inner mitochondrial membrane (IMM). While MPIS can also cause mitophagy induction through engagement of the NLRX1-RRBP1 pathway, we observed that DELE1-HRI and NLRX1-RRBP1 signaling were engaged independently upon MPIS. Surprisingly, our results suggest that in our cellular model the mitochondrial protease OMA1 was dispensable for DELE1 cleavage upon MPIS. Instead, we identified a key role for another mitochondrial protease, HtrA2, in mediating the cleavage of DELE1 into DELE1-S and DELE1-VS. Our data further suggest that DELE1 is likely cleaved into DELE1-S by HtrA2 in the cytosol, while the DELE1-VS form might be generated during halted translocation of the protein into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies HtrA2 as a critical protease involved in DELE1 processing. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The This is Marketing Podcast produced 24 podcasts in 2016 starting in April. Total Full Listens: 3230 Total 206 Listens (partial): 3910 Total Listens: 7140 Downloads Hits 206 Hits Bandwidth Average size /wp-content/uploads/2016/09/TIM23_mixdown.mp3 199 321 7.15 GB 14.07 MB /wp-content/uploads/2016/04/TIM1_mixdown.mp3 180 484 9.80 GB 15.11 MB /wp-content/uploads/2016/04/TIM3_mixdown.mp3 159 143 4.70 GB 15.95 MB /wp-content/uploads/2016/06/TIM9_mixdown.mp3 153 […]

In order to react to changes within their environment, plants developed a specific signaling network that enables the cells to convert external stimuli including light, abiotic and biotic stress as well as hormones into cellular signals. One example is the influx of calcium, a second messenger stored in apoplasts or internal reservoirs, into the cytosol. This causes changes in the calcium-ion-concentration within the cell that are recognized by specific sensors including Calmodulin and lead to the induction of a cellular signal response. Calcium signals do not only occur in the cytosol, but also appear within the nucleus, chloroplasts, mitochondria as well as peroxisoms (Bachs et al. 1992, Chigri et al. 2005, Kuhn et al. 2009, Dolze et al. 2013). The import of nuclear encoded proteins into the mitochondria is regulated by calcium and Calmodulin at level of the TIM23- and TIM22-complex. This study identified atTim23.2, the pore-forming component of the TIM23-complex, as a Calmodulin-binding protein. Pull-down-assays using Calmodulin-agarose revealed a specific and calcium-dependent binding. Furthermore, in silico analysis identified two potential Calmodulin-binding domains (CaMBD). Topology studies of atTim23.2 demonstrated that the proposed N-terminal CaMBD is located within the intermembrane space, the binding region within the first loops is located in the matrix of the mitochondria. Moreover, a topology of four transmembran domains of the protein could be shown. The recently in the mitochondria identified Calmodulin-like protein CML30 appeared to be a potential binding partner for atTim23.2. CML30 could be indeed detected in the intermembrane space of the mitochondria, but a direct interaction of the two proteins could not have been detected so far. Furthermore, using the split-ubiquitin system proved the ability of atTim23.2 to dimerize which might be responsible for the regulation of opening and closing of the importpore as it was already shown in S.cerevisiae. However, a correlation between the two functions of atTim23.2 to bind Calmodulin as well as to dimerize could not have been confirmed, yet. Nevertheless, the regulation of the pore via the calcium/Calmodulin signaling network could connect the import process of matrix proteins with the stress regulation of the cell.

Molecular chaperones of the Hsp70 class are essential for a number of cellular processes. The yeast mitochondrial Hsp70 chaperone Ssc1 plays an indispensable role for the mitochondrial biogenesis. As an essential component of the import motor of the TIM23 transolcase, Ssc1 drives the ATP-dependent translocation of proteins into the mitochondrial matrix. Moreover, it mediates the de novo folding and the assembly of several proteins in the mitochondrial matrix and prevents the formation of protein aggregates. Surprisingly, Ssc1 itself has a propensity to self-aggregate. Thus, it requires a helper protein, the chaperone Hep1 that prevents Ssc1 aggregation and maintains its structure and function. The mechanism of the protective function of Hep1 on Ssc1, however, is not understood. In the present study, the structural determinants of Ssc1 that make it prone to aggregation and the structural requirements of Ssc1 for its interaction with Hep1 were analysed and provided insights into the mechanism of prevention of Ssc1 aggregation by Hep1. The aggregation studies demonstrate that a variant of Ssc1 consisting of the ATPase domain and the subsequent interdomain linker aggregates in absence of Hep1. In contrast, the PBD and the ATPase domain alone are not prone to aggregation. Moreover, the interaction studies reveal that the aggregation-prone region seems to be the smallest entity within Ssc1 required for the interaction with Hep1. Taken together, the native Ssc1 adopts an aggregation-prone conformation, in which the ATPase domain with the interdomain linker has the propensity to aggregate. Hep1 binds to this aggregation-prone region and thereby counteracts the aggregation process and keeps the native Ssc1 in a functional and active state. Although Hsp70 chaperones are important for the biogenesis of a multitude of proteins, little is known about the biogenesis of these chaperones themselves. The present study reports on the analysis of the folding process of the mitochondrial Hsp70 chaperone Ssc1. In organello, in vivo and in vitro assays were established and then employed to study the de novo folding of Ssc1. Upon import into mitochondria, Ssc1 folds rapidly with the ATPase domain and the PBD adopting their structures independently of each other. Notably, the ATPase domain requires the presence of the interdomain linker for its folding, whereas the PBD folds without the linker. Moreover, in the absence of Hep1, the ATPase domain with the interdomain linker displays a severe folding defect, which indicates a role of Hep1 in the folding process of Ssc1. Apart from Hep1, none of the general mitochondrial chaperone systems seem to be important for the folding of Ssc1. Furthermore, the folding process of Ssc1 was reconstituted in vitro and the main steps of the folding pathway of Ssc1 were characterised. Hep1 and ATP/ADP are required and sufficient for the folding of Ssc1 into the native, catalytically active form. In an early step of folding, Hep1 interacts with the folding intermediate of Ssc1. This interaction induces conformational changes which allow binding of ATP/ADP. The binding of a nucleotide triggers Hep1 release and further folding of the intermediate into a native Ssc1. The present study provides the first direct evidence for the requirement of Hep1 for the folding of the Ssc1 chaperone. Thus, it demonstrates for the first time that the de novo folding of an Hsp70 chaperone depends on a specialized proteinaceous factor. In conclusion, Hep1 fulfils a dual chaperone function in the cell. It mediates the de novo folding of Ssc1 and maintains folded Ssc1 in a functional state during the ATPase cycle. Therefore, the Hep1 chaperone plays a crucial role for the protein biogenesis and homeostasis in mitochondria.

The vast majority of mitochondrial proteins are synthesized by the cytosolic ribosomes as precursor proteins which have to be transported into the organelle to reach their sites of function. The whole process of recognition, translocation, intra-mitochondrial sorting of and assembly of precursor proteins is achieved by the concerted action of different mitochondrial translocases. All proteins destined for the mitochondrial matrix and some inner membrane proteins are imported first by the TOM complex of the outer membrane and subsequently by the TIM23 complex of the inner membrane in an energy-driven process. The TIM23 complex was found to consist of ten components, conventionally divided into two sectors: membrane sector harbouring the translocation channel and the import motor on the matrix side of the membrane sector. In the first part of the present work, the two most recently discovered subunits of the TIM23 complex, Pam17 and Tim21 were characterized. A systematic characterization revealed that both of these non-essential subunits of the translocase are associated with Tim17-Tim23 core of the membrane sector of the TIM23 translocase. A functional connection between the two non-essential components was discovered. Results presented in this part showed that Pam17 and Tim21 modulate the functions of the TIM23 complex in an antagonistic manner. The second part of the work was directed towards understanding the motor sector of the translocase in terms of the regulated interaction between Tim44 and Ssc1. Previous studies on the Tim44:Ssc1 interaction were able to discern the steady-state properties of Tim44:Ssc1 interaction in organello and in vitro. However, due to the limitations of the techniques used, they were unable to shed light on the kinetics and dynamics of the process. The translocation event is a dynamic event with conformational cycling of the various components. Therefore, the kinetic components essential in defining the cycle of events in the motor sector were explored. A FRET based assay to analyze the Tim44:Ssc1 interaction in real time was developed. The same set of tools was also used to resolve the regions of the two proteins that determine their interaction. The substrate induced dissociation of Tim44:Ssc1 complex was found to be too slow to support a physiological rate of protein translocation. ATP-induced dissociation was observed to be fast enough to be physiologically relevant. The dissociation of Ssc1 from Tim44 occurred in a one step manner without Tim44 anchored conformational changes. Furthermore, peptide-array scanning of mitochondrial matrix proteins revealed that Ssc1 and Tim44 share complementary binding sites on the precursor proteins which could prevent backsliding of preproteins. The data support the Brownian ratchet model mediated translocation of preproteins into the mitochondrial matrix. The third part of the work aimed at dissecting the chaperone cycle of Ssc1 in the mitochondrial matrix, in terms of conformational changes and binding of co-chaperones. Using the FRET sensors developed, the inter-domain conformation and lid-base conformations of the PBD of Ssc1 could be investigated. Single particle FRET (SpFRET) analysis showed that in the ATP-bound form Ssc1 populates a homogeneous conformational state with respect to the inter-domain conformation and conformation of the lid to base of the PBD. On the contrary, in the ADP-bound state the conformation of the chaperone is heterogenous. Using the same sensors on bacterial homologue DnaK, specific differences in conformational distributions were observed. Furthermore, the active role of substrates in determining the inter-domain conformation and lid-closing was evident from the SpFRET based conformational analyses. Using ensemble time resolved FRET, the kinetics and dynamics of conformational changes along with binding of co-chaperones were explored. This provided a better understanding of the conformational dynamics of Ssc1 in the context of functional chaperone cycle in the mitochondrial matrix.

The vast majority of mitochondrial proteins are synthesized on cytosolic ribosomes in the form of precursor proteins and subsequently imported into mitochondria through the concerted action of the translocases present in the outer and the inner membrane. The TIM23 complex (translocase of the inner membrane) mediates translocation of precursor proteins across or their insertion into the mitochondrial inner membrane in a membrane potential and ATP-dependent manner. The TIM23 complex consists of eight essential subunits that can be assigned to two operationally defined parts: the membrane embedded protein conducting channel with the receptor and the import motor associated with the channel at the matrix side of the inner membrane. The present study was undertaken to gain insight into the dynamics of the TIM23 translocase during import of different types of preproteins. A previously uncharacterized protein component of the TIM23 translocase was identified and termed Tim21. Results presented in this study demonstrate that the TIM23 translocase switches between translocation mode that facilitates import of proteins into the matrix and insertion mode that allows lateral sorting of proteins into the lipid bilayer. The TIM23 translocase adopts different conformations in its various states of activity: when it was empty, when it inserted preproteins into the inner membrane and when it translocated preproteins targeted to the matrix. The interconversion of the TIM23 translocase between the functional states occurs primarily by conformational changes of the essential components, whereas non-essential components Tim21 and Pam17 are responsible for the fine tuning of these processes. A hypothesis that describes the behavior of the TIM23 translocase is presented.

Die Biogenese von Mitochondrien erfordert den Import von Präproteinen aus dem Cytosol in die mitochondrialen Subkompartimente. Der TIM23-Komplex der mitochondrialen Innenmembran ist für die Translokation von Präproteinen über die Innenmembran verantwortlich und vermittelt darüber hinaus die Insertion von Proteinen in die Innenmembran. Tim23 weist zwei funktionell unterscheidbare Domänen auf: Eine N-terminale hydrophile Rezeptordomäne im Intermembranraum und einen hydrophoben C-terminalen Bereich. Das phylogenetisch verwandte Tim17 ist ein sehr hydrophobes Protein, welches vier Transmembrandomänen ausbildet, die von zwei kurzen Enden im Intermembranraum flankiert werden. Die hydrophoben Bereiche von Tim17 und Tim23 bilden vermutlich den kanalbildenden Teil der Translokase. In der vorliegenden Arbeit wurde die Funktion von Tim17 bei der Translokation von Präproteinen über die Innenmembran untersucht. Es konnte eine kurze N-terminale Sequenz von 11 Aminosäureresten identifiziert werden, welche für die Funktionalität der TIM23-Translokase essentiell ist. Die Deletion dieser Sequenz beeinflusst die Integrität der bekannten Untereinheiten der TIM23-Translokase nicht, führt jedoch zu einer starken Beeinträchtigung der Translokation von Präproteinen über die mitochondriale Innenmembran. Durch gezielte Alanin-Punktmutagenese konnten zwei konservierte Aspartatreste in der Tim17-Sequenz identifiziert werden, welche für den Translokationsdefekt verantwortlich sind. Die Analyse weiterer Mutanten in Tim17 mit einzelnen oder wechselseitig ausgetauschten geladenen Aminosäureresten im Intermembranraum legen nahe, dass die konservierten negativen Ladungen in Tim17 mit den positiv geladenen Präsequenzen interagieren und dadurch die Translokation von Präproteinen durch den TIM23-Komplex regulieren. Diese Ergebnisse geben einen Einblick in eine Präprotein-abhängige Regulation der TIM23-Translokase über ein mögliches "Öffnen" und "Schließen" des Translokationskanals via Tim17. Die meisten Proteine der mitochondrialen Innenmembran, die als Präproteine mit mitochondrialen Präsequenzen im Cytosol synthetisiert werden, erreichen die Innenmembran auf einem von zwei alternativen Sortierungswegen: Dem "Stop-Transfer-Weg", auf dem Präproteine während der Translokation durch den TIM23-Komplex arretiert und lateral in die Innenmembran inseriert werden und dem Weg der "Konservativen Sortierung", auf dem die Proteine über Intermediate in der mitochondrialen Matrix in die Innenmembran inseriert werden. Folglich müssen diese Proteine entsprechende Sortierungssignale aufweisen, die entweder die laterale Membraninsertion (Stop-Transfer-Proteine) oder die die Translokation in die Matrix (konservativ sortierte Proteine) durch die TIM23-Translokase vermitteln. Das Sortierungsverhalten von mitochondrialen Innenmembranproteinen mit N-terminalen Präsequenzen, die zunächst für die initiale Translokation des N-Terminus der Proteine sorgen, wird von den Transmembrandomänen bestimmt. Um den Einfluss der Transmembrandomänen auf den Sortierungsweg zu untersuchen, wurden die entsprechenden Domänen von Stop-Transfer sortierten Proteinen und konservativ sortierten Proteinen wechselseitig ausgetauscht. In den chimären Proteinen bestimmten jeweils die eingeführten Transmembrandomänen das Sortierungsverhalten. Eine Untersuchung dieser Transmembrandomänen zeigte zwei systematische Unterschiede: Transmembrandomänen, die die konservative Sortierung vermitteln, weisen eine zumeist moderate Hydrophobizität auf und enthalten zumeist Prolinreste. Dagegen sind Stop-Transfer vermittelnde Transmembrandomänen typischerweise stärker hydrophob und frei von Prolinresten. Die Einführung von Prolinresten in die Transmembrandomänen von ursprünglich Stop-Transfer sortierten Proteinen führte zu deren Translokation in die Matrix. Umgekehrt führte die Mutagenese von Prolinresten in Transmembrandomänen ursprünglich konservativ sortierter Proteine zu deren Arretierung in der Innenmembran. Die Anwesenheit von Prolinresten in den Transmembrandomänen bestimmt demnach den Sortierungsweg dieser Innenmembranproteine. Zukünftige Studien werden zeigen, wie diese Sortierungssignale, welche eventuell eine von Prolinresten gebrochene hydrophobe Helix darstellen, von der TIM23-Translokase erkannt und entsprechend umgesetzt werden. Die Bedeutung von Prolinresten in Transmembrandomänen von konservativ sortierten Proteinen konnte durch Mutagenese sowohl in vitro als auch in vivo gezeigt werden. Diese Erkenntnis sollte sowohl in Vorhersagen von Proteinsortierungswegen als auch bei der zukünftigen Entwicklung mitochondrialer Proteine für gentherapeutische Ansätze zur Behandlung mitochondrialer Erkrankungen berücksichtigt werden.

Mitochondria are essential cellular organelles of eukaryotic organisms, which import most of their proteinaceous constituents from the cytoplasm. Two mitochondrial membranes contain different translocation machineries which are involved in the import and proper sorting of mitochondrial precursor proteins. The TIM22 translocase in the inner mitochondrial membrane mediates the import of polytopic proteins into this membrane. In addition to the membrane integrated components Tim22 and Tim54, the TIM22 translocase possesses components in the intermembrane space, termed Tim9 and Tim10. In the present study, the tim9 and tim10 genes of the TIM22 translocase of N. crassa were identified. The structural and functional characteristics of the corresponding gene products, the Tim9 and Tim10 proteins, were examined. Tim9 was demonstrated to be an essential protein. The Tim9 and Tim10 proteins were shown to build a 70-80 kDa heterohexameric complex in the mitochondrial intermembrane space. The isolated Tim9•Tim10 complex had the same oligomeric structure as the native one, and it proved fully functional in interacting in vitro with its physiological substrate, the ADP/ATP carrier (AAC). Peptide library screens were performed to determine the structural determinants of the substrates that are recognised by the Tim9•Tim10 complex. Efficient binding to the regions covering residues of the hydrophobic membrane spanning domains and of the connecting hydrophilic loops was observed. In this way, Tim9 and Tim10 proteins interact with their substrates, while the hydrophobic regions of the substrates are still present in the TOM complex and thereby protected from the aqueous environment of the intermembrane space compartment. Furthermore, when enclosed into proteoliposomes containing the reconstituted TOM complex, Tim9•Tim10 complex specifically promoted the translocation of the AAC precursor. Hence, the Tim9•Tim10 complex and the TOM complex are both necessary and sufficient to facilitate translocation of carrier proteins across the outer mitochondrial membrane. Finally, peptide screens and chemical cross-linking experiments were used to identify the precursor of N. crassa Tim23 protein as a novel substrate of the Tim9•Tim10 complex.

Wed, 12 May 2004 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/2330/ https://edoc.ub.uni-muenchen.de/2330/1/Mokranjac_Dejana.pdf Mokranjac, Dejana ddc:570, ddc:500, Fakultät für Biologi

The molecular basis of the Mohr-Tranebjaerg syndrome: A structural and functional analysis of the proteins DDP1 and Tim13 Mohr-Tranebjaerg syndrome is a mitochondrial disorder caused by a defects in the biogenesis of the human TIM23 translocase Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organised in hetero-oligomeric complexes in the mitochondrial intermembrane space (IMS). The TIM8-13 complex assists the import of Tim23, the major component of the translocase for matrix-targeted proteins. Mutations in DDP1/TIMM8A, the gene encoding the human homolog of Tim8, cause the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder. This work shows that DDP1 and human Tim13 are zinc binding proteins which together form a 70 kDa complex in the intermembrane space of human mitochondria. Similar to yeast, the human DDP1-hTim13 complex facilitates import of yeast and human Tim23. It has been additionally analysed the structural and functional consequences of a MTS-missense mutation (C66W) directly affecting the conserved Cys4 metal binding motif. In this connection the C66W mutation impairs the ability to bind zinc. As a consequence, the mutated DDP1 loses its ability to assemble into a hetero-oligomeric complex with its partner protein human Tim13. Thus, it was suggested that an assembly defect of DDP1 is the molecular basis of Mohr-Tranebjaerg syndrome in patients carrying the C66W mutation.