טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentChen Lesnik
SubjectRole of Cytosolic Chaperones in Co-Translational Targeting
to Yeast Mitochondria
DepartmentDepartment of Biology
Supervisor Professor Arava Yoav
Full Thesis textFull thesis text - English Version


Abstract

The eukaryotic cell contains many proteins that perform diverse functions at different compartments. To achieve this complexity, newly synthesized proteins need to be targeted to their proper destination. Multiple modes of protein targeting have evolved. We are focusing on one mode in which the protein is targeted while being translated. We previously found that a receptor on the mitochondrial outer membrane (Tom20) induces mRNA association with the mitochondria, presumably by binding the Mitochondrial Targeting Sequence of the nascent polypeptide- mRNA- ribosome complex. In this work, we show that a protein chaperone (Ssa1) is important for the localization of mRNAs to the mitochondria, in particular those encoding hydrophobic proteins and proteins with relatively high number of aromatic amino acids. Moreover, Ssa1 is able to improve mitochondrial localization of mRNA that is mutated in its MTS. This suggests that the role of Ssa1 does not depend on the MTS, and hence may not require Tom20. This suggests that a co-translational targeting process exists also for proteins that are destined to the mitochondria.

We also explored the role of a ribosome- associated complex (NAC) in this process. NAC is a conserved heterodimer that was shown to affect association of ribosomes with mitochondria and to support co-translational import into this compartment. We performed a protein complementation screen for proteins that interact with each one of the NAC subunits. We found that the mitochondrial outer membrane protein Om14 is a positive partner that interacts with both subunits of NAC. Om14 appeared to interact with NAC in all eight different types of screens we performed. Co- immunoprecipitation analyses confirmed these results. Furthermore, mitochondrial fraction from cells deleted of Om14 had significantly reduced levels of NAC and ribosomes. Complementarily, ribosomes isolated from NAC- deleted cells had reduced Om14 association. We also tested the biologic significance of NAC-Om14 interaction through import assays into mitochondria. Our results show that Om14- deleted mitochondria have reduced co-translational import efficiency, and this role in import is exerted through NAC. Thus, Om14 is a receptor for ribosome-associated NAC, thereby coordinating localized translation and import to the mitochondria.

We also examined the role of NAC in co-translational targeting to human mitochondria by testing the mRNA localization of mitochondrial mRNA markers in either WT or NAC- depleted cells. Our preliminary results show that NAC knockdown affects mRNA localization to mitochondria in an unknown mechanism. Further experiments are necessary to characterize NAC’s role in human cells.