Identifizierung und Charakterisierung neuer Faktoren der zytosolischen Fe-S Proteinbiogenese

Iron sulfur (Fe-S) clusters are important cofactors of numerous proteins and are present in all known forms of life. Fe-S proteins have various functions in electron transport, enzyme catalysis and gene regulation. Moreover, recently Fe-S cluster-containing proteins with a function in essential cellular processes such as DNA replication, DNA repair, transcription and ribosome function were detected. The synthesis of the Fe-S clusters and their subsequent insertion into apoproteins require three complex proteinaceous machineries in eukaryotes that are localized in the mitochondria and the cytosol. The maturation of cytosolic and nuclear Fe-S proteins is accomplished by the cooperation of the mitochondrial iron sulfur cluster (ISC) assembly machinery, the ISC export machinery and the cytosolic iron sulfur protein assembly (CIA) machinery. The CIA machinery consists of eight known proteins but their interactions with each other are not fully understood. Moreover it is likely that even more CIA proteins are involved in this complex biosynthetic process. In the first part of my thesis the interactions of previously known CIA proteins were analyzed in the model organism Saccharomyces cerevisiae. By performing a systematic CIA protein interaction analysis it was shown that the individual CIA factors do not assemble a supercomplex. They rather can be divided into three functional subcomplexes that assist different stages in biogenesis of cytosolic and nuclear Fe-S proteins. The scaffold protein complex consisting of Cfd1-Nbp35 is required for the assembly of a [4Fe-4S] cluster in the early phase of biogenesis. Electrons required for this reaction are provided by the CIA subcomplex Dre2-Tah18. In the late phase of cytosolic Fe-S protein biogenesis the newly assembled Fe-S cluster is transferred from the scaffold protein complex onto apoproteins. This step is performed by the so-called "CIA targeting complex" consisting of Cia1, Cia2 and Met18. The CIA factor Nar1 fulfills an intermediary, so far not well-defined function and interacts with both the early and the late parts of the CIA machinery. In the second part of my thesis the essential proteins Yae1 and Lto1 were discovered and characterized as new components of the CIA machinery. These two proteins are highly conserved and form a complex that is specifically required under aerobic conditions. In a proteome-wide interaction analysis Yae1 and Lto1 were identified as binding partners of the CIA targeting complex. Moreover, a previous systematic interaction analysis has shown an association of Yae1-Lto1 with the Fe-S protein Rli1. However, a functional connection of these proteins to the CIA machinery has not been reported until now. It was shown in this work that the depletion of Yae1 or Lto1 results in a maturation defect of the essential Fe-S protein Rli1 but surprisingly no other cytosolic or nuclear target proteins were affected. Additionally, by setting up a new technology I showed that the cofactors were stably associated with Rli1 when Yae1 was rapidly degraded, confirming that this protein is not a stabilization factor of the two Fe-S clusters. These findings suggest that Yae1 and Lto1 are highly specific maturation factors for Rli1. The Yae1-Lto1 complex is conserved in eukaryotes and can be functionally replaced by the human YAE1D1-ORAOV1 complex in yeast. This work shows that certain cytosolic Fe-S proteins such as Rli1 obviously do not only depend on the general CIA machinery for the maturation of their Fe-S clusters, but also on the function of other more specialized CIA components. A comparable substrate specificity as observed for these two new CIA factors was described recently for the human CIA factor CIA2A, which is solely responsible for the maturation of the cytosolic Fe-S protein IRP1. It has to be clarified whether additional CIA factors exist that support the dedicated maturation of eukaryotic Fe-S proteins. Furthermore, future studies will have to address the molecular mechanism by which such highly specific factors conduct the insertion of the Fe-S clusters into target proteins.