Dokument

Zusammenfassung:
Eisen ist ein essentielles Spurenelement mit entscheidenden Funktionen in einer Vielzahl an metabolischen Prozessen wie der mitochondrialen Atmung, Aminosäure- und Nukleotid-Synthese, Ribosomen-Assemblierung, DNA-Replikation und DNA-Reparatur. Jedoch sind erhöhte zelluläre Eisenkonzentrationen toxisch für die Zelle. Daher ist eine exakte Regulation des intrazellulären Transports und der Erfassung des Eisenstatus erforderlich, um sowohl Eisenmangel als auch Eisenüberschuss zu vermeiden. Die vorliegende Arbeit widmet sich deshalb dem Mechanismus der mitochondrialen Eisenaufnahme und der Adaption an erhöhte Eisenmengen. Mitochondrien sind essentielle Organellen, die unter anderem eine entscheidende Rolle in der Eisen-Schwefel-(Fe/S)-Cluster- und in der Häm-Biogenese spielen. Aufgrund dieser Stoffwechselleistungen konsumieren Mitochondrien den größten Anteil des in die Zelle aufgenommenen Eisens. Frühere Studien konnten die mitochondrialen Carrier-Proteine Mrs3 und Mrs4 als mitochondriale Eisentransporter charakterisieren. Allerdings ist die simultane Deletion beider Gene für die Hefezellen nicht letal. Da die Fe/S-Cluster Biogenese essentiell ist, lässt dies auf einen weiteren Carrier schließen, der ebenfalls in der Lage ist, Eisenionen zu transportieren. Ein vielversprechender Kandidat ist der mitochondriale Carrier Rim2. Auch wenn dieser ursprünglich als Pyrimidintransporter charakterisiert wurde, häufen sich die Hinweise auf eine zusätzliche Funktion in der Eisenaufnahme. Dabei stellt sich die Frage, inwiefern sich die gut dokumentierte Transportfunktion für Pyrimidine mit einer möglichen Rolle im Eisentransport vereinen ließe. Im ersten Teil dieser Arbeit konnte gezeigt werden, dass die zusätzliche Deletion von RIM2 die in mrs3/4Δ Zellen vorhandene Defekte im Wachstum und in der Biosynthese von Eisen-abhängigen Proteinen verstärkt. Dagegen rettet die Überexpression von RIM2 in mrs3/4Δ Zellen die Maturierung der Fe/S-Proteine und der Häm-Synthese auf Wildtypniveau. Demzufolge sind hohe Konzentrationen von Rim2 ausreichend, um die mitochondriale Eisenversorgung in mrs3/4Δ Zellen wiederherzustellen. Der direkte biochemische Nachweis für eine Transportfunktion von Rim2 wurde durch in vitro Transportexperimente mit sog. submitochondrialen Partikeln in Zusammenarbeit mit der Arbeitsgruppe Dr. Wiesenberger (Wien) geliefert. Zusammengenommen zeigen die in vivo, in organello und in vitro Experimente, dass Rim2 als mitochondrialer Carrier den obligatorischen Co-Transport von Pyrimidinen und divalenten Metallionen einschließlich Fe2+ vermittelt. Diese Modelvorstel-lung erklärt, wie die mitochondriale Eisenaufnahme in mrs3/4Δ Zellen gesichert werden kann und wie Rim2 als „high copy suppressor“ von mrs3/4Δ Zellen fungiert. Eine Funktion des Rim2 in der mitochondrialen Eisenhomöostase unter normalen physiologischen Bedingungen, d.h. in Anwesenheit von Mrs3 und Mrs4, ist jedoch vernachlässigbar. Der zweite Teil dieser Arbeit konzentrierte sich auf die Charakterisierung des Sensors für hohe Eisenkonzentrationen, Yap5, der eine zentrale Rolle bei der Anpassung an toxische Eisenmengen in S. cerevisiae spielt. Bei Eisenüberschuss aktiviert der Transkriptionsfaktor Yap5 die Expression von CCC1, dem einzigen bekannten vakuolären Eisenimporter, was zur Speicherung von Eisen in der Vakuole und damit zur Vermeidung einer toxischen Eisenakkumulation im Cytosol führt. Wie Yap5 biochemisch den Eisenstatus der Zelle erfasst, ist allerdings unklar. Zunächst konnte die direkte Bindung von radioaktivem Eisen (55Fe) an die Aktivator-Domäne von Yap5 (tYap5) in vivo nachgewiesen werden. Zirkular-dichroismus-, Elektronenspinresonanz- und Mößbauer-Spektroskopie des rekombinanten tYap5 belegten die Bindung eines [2Fe-2S]-Clusters an tYap5 nach chemischer Rekonsti-tution. Dieser [2Fe-2S]-Cluster wird durch die N-terminale Cystein-reiche-Domäne (n-CRD) in der Aktivator-Domäne koordiniert und ist entscheidend für die transkriptionelle Aktivität. Des Weiteren induziert die Fe/S-Cluster-Bindung an tYap5 eine Konformationsänderung, die möglicherweise geeignet ist, die Transkriptionsaktivität von Yap5 zu modulieren. Zusätzlich bindet Yap5 in vitro einen zweiten Fe/S-Cofaktor an der C-terminalen CRD, der nicht an der Regulation der transkriptionellen Aktivität von Yap5 in vivo beteiligt ist. Dagegen ist das Fe/S-Cluster-Bindungsmotiv innerhalb der regulatorischen n-CRD von Yap5 in mehreren Pilzarten konserviert, u.a. auch im Aspergillus Transkriptionsfaktor HapX. Daher könnte dieses Motiv als konservierte Bindungsstelle für einen sensorischen Fe/S-Cluster in der eukaryotischen Stressantwort fungieren. Zusammengefasst hat diese Arbeit an Yap5 grundlegende neue Einblicke in den transkriptionellen Mechanismus der Erfassung und Regulation des Eisenstatus in Pilzen geliefert. Yap5 konnte somit als erster eukaryotischer Transkriptionsfaktor, der einen Fe/S-Cluster als Sensor für intrazelluläre Eisenkonzentrationen nutzt, charakterisiert werden.

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