Biogenese von Proteinen mit mehreren Transmembranhelices in der mitochondrialen Aussenmembran
Zellbiologie
Zusammenfassung der Projektergebnisse
Mitochondria are essential eukaryotic organelles for energy production as well as various cellular activities. The maintenance of proper mitochondrial function is achieved by proteins located in different mitochondrial sub-compartments. Proteins embedded in the mitochondrial outer membranes (MOM) with several transmembrane segments are defined as multi-span proteins and considerable efforts have been made to understand their biogenesis. Newly synthesized precursors of these proteins are believed to be recognized by the receptor Tom70 on the surface of mitochondria. However, the precise contribution of Tom70 to this process is poorly understood. In this project, we analyzed the involvement of the Tom70 cytosolic domain in the recognition of different substrates. we found that Tom70 could directly interact with multi-span MOM proteins and function as a docking site for precursorbound chaperones. The relative importance of the two functions of Tom70 varies from substrate to substrate. After the initial recognition, multi-span outer membrane precursor proteins are passed on to the MIM complex which further facilitate their integration into the MOM. Nevertheless, little is known about the targeting signal within such substrates and the involvement of other potential factors in their biogenesis. Interestingly, another report suggested that the integration of multi-span OMM proteins is only lipid-regulated and can occur spontaneously without the help of any import factors. In our project, we tried to reconcile this apparent discrepancy and obtain a deep knowledge of the biogenesis of these proteins using the yeast multi-span OMM protein Om14 as a model protein. We first examined the targeting behavior of different truncation variants and found out that multiple targeting signals collectively contributes to the perfect mitochondrial targeting specificity. Next, we employed a specific import assay to test the effect of different elements on the insertion efficiency of Om14. The results suggest that none of the cytosolic exposed domains of proteinaceous factors is essential for the Om14 biogenesis process. In contrast, we could demonstrate that both Mim1 and Porin are involved in the optimal membrane integration of Om14. In addition, we analyzed the influence of the unfolding of newly synthesized Om14, the hydrophobicity of its transmembrane segments, and an increased membrane fluidity on the efficiency of its membrane integration. We observed that unfolding of the substrate protein and elevated temperature increased the import efficiency of Om14 whereas reduced hydrophobicity of the second transmembrane segment reduced it. Collectively, our results provide a novel insight into the biogenesis of the multi-span OMM protein Om14, suggesting the coexistence of many alternative routes in which both proteinaceous factors and membrane behavior contribute to varying degrees to the combined insertion efficiency.
Projektbezogene Publikationen (Auswahl)
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(2020) Biogenesis pathways of α-helical mitochondrial outer membrane proteins. Biol. Chem., 401, 677-686
Drwesh, L., and D. Rapaport
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(2021) Cnm1 mediates nucleus-mitochondria contact site formation in response to phospholipid levels. J. Cell Biol., 220, e202104100
Eisenberg-Bord, M., N. Zung, J. Collado, L. Drwesh, E. Fenech, A. Fadel, N. Dezorella, Y. Bykov, D. Rapaport, R. Fernandez Busnadiego, and M. Schuldiner
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(2021) The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress. Cell Rep., 35, 108936
Backes, S., Y.S. Bykov, T. Flohr, M. Räschle, J. Zhou, S. Lenhard, L. Krämer, T. Mühlhaus, C. Bibi, C. Jann, J.D. Smith, L.M. Steinmetz, D. Rapaport, Z. Storchová, M. Schuldiner, F. Boos, and J.M. Herrmann
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(2022) The multi-factor modulated biogenesis of the mitochondrial multi-span protein Om14. J. Cell Biol., 221, e202112030
Zhou, J., M. Jung, K.S. Dimmer, and D. Rapaport
