Protein integration into the mitochondrial outer membrane
Final Report Abstract
Biogenesis of the mitochondrial outer membrane (MOM) involves integration of newly synthesized proteins into the lipid bilayer. Despite ever increasing evidence for the biological significance of these proteins, our understanding of their biogenesis is scarce. The general objective of this project was to fully define the biological processes and molecular mechanisms that underlie the biogenesis of MOM proteins. We concentrated on two groups of proteins: those that span the membrane once, exposing domains to both the cytosol and the mitochondrial intermembrane space and the group of protein that traverse the membrane as multiple helical segments. In the first part of the project we searched for proteins that mediate the biogenesis of the single-span protein Mim1. Based on our results we suggest that newly synthesized Mim1 molecules are recognized in the cytosol by the co-chaperone J-protein Djp1, which then also engages the corresponding Hsp70 chaperone. Next, the mitochondrial precursor protein and its chaperones associate with the import receptor Tom70. The substrate is released from the chaperones and gets inserted into the outer membrane. Decoding the mechanism of the down-stream membrane integration awaits future studies. In another part of the project we investigated the import mechanism of the multispan protein Om14. To that end, we performed complementary high-throughput visual and growth screens in yeast. Cardiolipin synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4 and Om14 in mitochondria from cells deleted for CRD1. Moreover, organelles lacking Crd1 had a reduced ability to import in vitro newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain cardiolipin (CL) molecules. Hence, our findings suggest CL as an important component in the biogenesis of MOM multispan proteins. In a complementary approach to obtain a detailed understanding of the import of multispan OM proteins, we attempted to functionally reconstitute the MIM complex into lipid vesicles. However, despite many efforts while using S. cerevisiae, N. crassa and E. coli cells, we were not able to obtain sufficient amounts of both components (Mim1 and Mim2) in pure form. These technical difficulties prevented us from achieving this goal. Taken together, our project brought new insights into the biogenesis of proteins residing in the mitochondrial outer membrane. Despite this progress, the molecular mechanism of the various import pathways is far from being understood and should be addressed in future studies.
Publications
- (2013) The role of Djp1 in import of the mitochondrial protein Mim1 demonstrates specificity between a co-chaperone and its substrate protein. Mol. Cell. Biol., 33, 4083-4094
Papić, D., Y. Elbaz-Alon, S.N. Koerdt, K. Leopold, D. Worm, M. Jung, M. Schuldiner, and D. Rapaport
(See online at https://doi.org/10.1128/MCB.00227-13) - (2014) Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1. Cell Death Dis., 5, e1180
Burbulla, L., J. Fitzgerald, K. Stegen, J. Westermeier, A.-K. Thost, H. Kato, D. Mokranjac, J. Sauerwald, L. Martins, D. Woitalla, D. Rapaport, O. Riess, T. Proikas- Cezanne, T. Rasse, and R. Krueger
(See online at https://doi.org/10.1038/cddis.2014.103) - (2015) Genome-wide screens in yeast highlight a role for cardiolipin in biogenesis of mitochondrial outer membrane multispan proteins. Mol. Cell. Biol. 35, 3200-3211
Sauerwald, J., T. Jores, M. Eisenberg-Bord, S.G. Chuartzman, M. Schuldiner, and D. Rapaport
(See online at https://doi.org/10.1128/MCB.00107-15)
