Podcasts about ssc1

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.

Folding of newly synthesized proteins is an essential part of protein biosynthesis and misfolding can result in protein aggregation which can also lead to several severe diseases. Protein folding is a highly heterogeneous process and rarely populated intermediate states may play an important role. Single-molecule techniques are ideally suited to resolve these heterogeneities. In this thesis, I have employed a variety of single-molecule fluorescence spectroscopy techniques to study protein folding using model systems on different levels of complexity. The acidic compact state (A state) of Myo- globin is used as a model system of a protein folding intermediate and is studied by a combination of molecular dynamics (MD) simulations and several fluorescence spectroscopic techniques. Using two-focus fluorescence correlation spectroscopy (FCS), it is shown that the A state is less compact than the native state of myoglobin, but not as expanded as the fully unfolded state. The analysis of exposed hydrophobic regions in the acidic structures generated by the MD simulations reveals poten- tial candidates involved in the aggregation processes of myoglobin in the acidic compact state. These results contribute to the understanding of disease-related fibril formation which may lead ultimately to better treatments for these diseases. A huge machinery of specialized proteins, the molecular chaperones, has evolved to assist protein folding in the cell. Using single molecule fluorescence spectroscopy, I have studied several members of this machinery. Single-pair fluorescence resonance energy transfer (spFRET) experiments probed the conformation of the mitochondrial heat shock protein 70 (Hsp70), Ssc1, in different stages along its functional cycle. Ssc1 has a very defined conformation in the ATP state with closely docked domains but shows significantly more heterogeneity in the presence of ADP. This heterogeneity is due to binding and release of ADP. The nucleotide-free state has less inter-domain contacts than the ATP or ADP-bound states. However, the addition of a substrate protein decreases the interaction between the domains even further simultaneously closing the substrate binding lid, showing that substrate binding plays an active role in the remodeling of Ssc1. This behavior is strikingly different than in DnaK, the major bacterial Hsp70. In DnaK, complete domain undocking in the presence of ADP was observed, followed by a slight re-compaction upon substrate binding. These differences may reflect tuning of Ssc1 to meet specific functions, i.e. protein import into mitochondria, in addition to protein folding. Ssc1 requires the assistance of several cofactors depending on the specific task at hand. The results of spFRET experiments suggest that the cofactors modulate the conformation of Ssc1 to enable it to perform tasks as different as protein import and protein folding. Downstream of Hsp70 in the chaperone network, the GroEL/ES complex is a highly specialized molecular machine that is essential for folding of a large subset of proteins. The criteria that distin- guish proteins requiring the assistance of GroEL are not completely understood yet. It is shown here that GroEL plays an active role in the folding of double-mutant maltose binding protein (DM-MBP). DM-MBP assumes a kinetically trapped intermediate state when folding spontaneously, and GroEL rescues DM-MBP by the introduction of entropic constraints. These findings suggest that proteins with a tendency to populate kinetically trapped intermediates require GroEL assistance for folding. The capacity of GroEL to rescue proteins from such folding traps may explain the unique role of GroEL within the cellular chaperone machinery.

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.

6 Literatur Ziel der vorliegenden Arbeit war es, die Lokalisation und Funktion des Hsp70- Homologen, Ecm10, zu klären. Ecm10 wurde als drittes Hsp70-Protein neben Ssc1 und Ssq1 in der mitochondrialen Matrix lokalisiert. Es besteht eine hohe Sequenzähnlichkeit zwischen Ecm10 und Ssc1, woraus eine ähnliche Funktionsweise resultiert. Die teilweise Funktionsüberlappung konnte in ssc1-3 ∆ecm10-Zellen durch einen synthetischen Wachstumsphänotyp experimentell nachgewiesen werden. Ecm10, das kein abundantes Protein ist, konnte jedoch selbst nach Überexpression Ssc1 nicht funktionell ersetzen. Da keine endogenen Substrate für Ecm10 bekannt sind, wurde die Funktion von Ecm10 im Vergleich zu Ssc1 in vitro analysiert. Ecm10 kann bei Überexpression den Proteinimport in die Matrix auch ohne funktionelles Ssc1 vollständig wiederherstellen. Wie Ssc1 bindet Ecm10 über eine Wechselwirkung mit Tim44 an die zu translozierende Polypeptidkette. Weiterhin verfügt es über eine ATPase-Domäne, deren Aktivität über die Wechselwirkung mit Mge1 reguliert wird. Im Gegensatz zu Ssc1 scheint Ecm10 jedoch nur über eine verminderte Faltungsaktivität zu verfügen, wobei nicht geklärt ist, ob diese durch eine eingeschränkte Wechselwirkung mit dem Cochaperon Mdj1 erklärt werden kann. Welche Rolle die gezeigten Funktionalitäten unter physiologischen Bedingungen für Ecm10 spielen, bleibt weiterhin offen. Des Weiteren wurde die Sortierung polytoper Membranproteine der mitochondrialen Innenmembran untersucht. Dazu wurde auf zwei bitope Beispielproteine, Mrs2 und Yta10, zurückgegriffen. Beide verfügen über jeweils eine negativ geladene, von zwei Transmembrandomänen eingerahmte Intermembranraumdomäne, die jedoch unterschiedlich groß ist. Es konnte gezeigt werden, dass beide Proteine dem konservativen Sortierungsweg folgen, in dessen Verlauf ein lösliches Sortierungsintermediat von der Matrix aus in die Innenmembran inseriert wird. Dabei ist der Insertions- oder Exportschritt aus der Matrix im Vergleich zum Import in die Matrix in höherem Maße abhängig vom Membranpotential über die Innenmembran. In beiden Fällen erfolgte die Sortierung unabhängig von den bisher bekannten Insertionsfaktoren Oxa1 und Mba1, was auf die Existenz weiterer Insertionsfaktoren deuten könnte. Die Untersuchung der Ladungsverteilung innerhalb der Intermembranraumdomänen verschiedenster mitochondrialer Innenmembranproteine ergab eine eindeutige Bevorzugung von sauren Resten, was auf einen allgemeinen Sortierungsweg für solche Proteine hindeutet, die aus bakteriellen Vorläufern abgeleitet wurden.