Deletion of Mgr2p Affects the Gating Behavior of the TIM23 Complex

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology to investigate the role of Mgr2p on the channel activity of TIM23. Deletion of Mgr2p decreased normal channel frequency and increased occurrence of a residual TIM23 activity. The residual channel lacked gating transitions but remained sensitive to synthetic import signal peptides. Similarly, a G145L mutation in Tim23p displaced Mgr2p from the import complex leading to gating impairment. These results suggest that Mgr2p regulates the gating behavior of the TIM23 channel.Peer reviewe

Deletion of Mgr2p Affects the Gating Behavior of the TIM23 Complex

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology to investigate the role of Mgr2p on the channel activity of TIM23. Deletion of Mgr2p decreased normal channel frequency and increased occurrence of a residual TIM23 activity. The residual channel lacked gating transitions but remained sensitive to synthetic import signal peptides. Similarly, a G145L mutation in Tim23p displaced Mgr2p from the import complex leading to gating impairment. These results suggest that Mgr2p regulates the gating behavior of the TIM23 channel

Biogenesis of Mitochondrial Signal-anchored Proteins - From Early Cytosolic Events to their Membranal Integration

Mitochondrien beherbergen in ihrer äußeren Membran (OM) Proteine verschiedener Topologien. In dieser Arbeit beschäftigte ich mich mit einer Proteinfamilie, die als signalanchored (SA) Proteine bezeichnet werden. Diese Familie besteht aus Proteinen, die die Membran mit ihrer α-helikalen Transmembrandomäne am N-Terminus einfach durchspannen, während ein großer Teil der C-terminalen Domäne zum Zytosol hin freiliegt. Wie die übrigen mitochondrialen OM-Proteine werden auch SA-Proteine im Zellkern kodiert, von zytosolischen Ribosomen translatiert, anschließend zum Zielorganell geleitet und in die Lipiddoppelschicht eingebettet. Damit die Mitochondrien ihre biologische Funktionen erfüllen können, ist es entscheidend, dass solche Proteine richtig integriert und ausgerichtet werden. Doch wie SA-Proteine in das Zielorganell importiert werden und welche vorherigen Ereignisse im Zytosol dazu benötigt werden, ist nahezu unbekannt. Zielsetzung dieser Arbeit war, die Schritte der Biogenese von SA-Proteinen, beginnend mit ihrer Synthese im Zytosol, bis hin zu ihrer Erkennung an der mitochondrialen Oberfläche zu entschlüsseln. Zu diesem Zweck habe ich eine Reihe von in vivo, in organello und in vitro Assays durchgeführt, bei denen ich verschiedene mitochondriale SA-Proteine als Modellproteine verwendet habe. Ich fand heraus, dass der MIM-Komplex für die Insertion des SA-Proteins Msp1 in die OM erforderlich ist, während andere Proteine aus dieser Kategorie über andere Wege inseriert werden. Dies deutete darauf hin, dass Proteine derselben Kategorie nicht unbedingt demselben Importmechanismus folgen, sondern in unterschiedlichem Maße auf verschiedene Importfaktoren angewiesen sind. Um die früheren zytosolischen Ereignisse aufzuklären, die für die Aufrechterhaltung der neu-synthetisierten Proteine in einer importkompetenten Konformation wesentlich sind, habe ich den Einfluss einiger zytosolischen Chaperone untersucht. Ich fand heraus, dass einige Chaperone aus verschiedenen Familien mit neu-synthetisierten SA-Proteinen über ihre hydrophoben Segmente interagieren. Außerdem konnte ich zeigen, dass solche Wechselwirkungen nicht nur entscheidend für die Stabilität der SA-Proteine im Zytosol sind, sondern auch für deren Import in das Organell und ihre optimale Ausrichtung benötigt werden. Als nächstes untersuchte ich die Bedeutung des Zusammenspiels zwischen den zytosolischen Chaperonen und den mitochondrialen Rezeptoren für die Biogenese von SAProteinen. Meine Ergebnisse deuteten auf eine Rolle der TOM-Komplex-Rezeptoren in Zusammenarbeit mit einigen Hsp70 und Hsp40 Chaperonen bei der Erkennung und dem Einbau von SA-Proteinen hin. Zusammengefasst bieten meine Ergebnisse neue Einblicke in die früheren zytosolischen Ereignisse bei der Biogenese von SA-Proteinen, von ihrer Synthese bis zu ihrer Erkennung an der mitochondrialen Oberfläche.Mitochondria harbour proteins with different topologies in their outer membrane (OM). In this study, I focus on one topological protein category, known as signal-anchored (SA) proteins. This family consists of proteins that span the membrane once via a single helical TMS located at their N-terminus, exposing a large C-terminal domain towards the cytosol. Like all mitochondrial OM proteins, SA proteins are encoded by nuclear DNA, translated by cytosolic ribosomes, and then are targeted to the organelle. Assuring proper and efficient targeting of such proteins is crucial for maintenance of mitochondrial biological function. Despite their importance, our understanding of the import routes that SA proteins follow including the early cytosolic events is scarce. In this study, I aimed to unravel the biogenesis steps of SA proteins following their synthesis in the cytosol until their recognition at the mitochondrial surface. To this end, I have applied a wide set of in vivo, in organello, and in vitro assays using various mitochondrial SA substrates as model proteins. I found that the MIM complex is required for the membrane insertion of the SA quality control protein Msp1, while other proteins from this category appear to follow different routes. These findings suggest that proteins from the same category may not necessarily follow the same pathway, but rather rely on different import factors to varying degrees. To shed light on the early cytosolic events that are essential for maintaining the newly synthesized SA proteins in an import competent conformation, I analysed the involvement of some cytosolic chaperones in their early biogenesis stages. I found that chaperones from distinctive families interact with newly synthesized SA proteins through the hydrophobic segments of the latter. I further could show that such interactions are not only crucial for keeping SA proteins stable in the cytosol, but also for their optimal targeting and import into the organelle. Next, I investigated the implication of the interplay between cytosolic chaperones and mitochondrial receptors on the biogenesis of SA proteins. My findings suggest a role of the TOM complex receptors in collaboration with the Hsp40 and Hsp70 chaperones in mediating the recognition and the insertion of SA proteins. Overall, my findings provide new insights into the early cytosolic events in the biogenesis of SA proteins following their synthesis until their recognition at the mitochondrial surface

A network of cytosolic (co)chaperones promotes the biogenesis of mitochondrial signal-anchored outer membrane proteins

Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins