Insights into mitochondrial presequence and carrier import pathways

by Ridhima Gomkale
Doctoral thesis
Date of Examination:2018-11-12
Date of issue:2020-10-20
Advisor:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Holger Stark
Referee:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Stefan Jakobs
Referee:Dr. Alexander Stein
Persistent Address: http://dx.doi.org/10.53846/goediss-8264

 

 

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Abstract

English

The mitochondria of yeast Saccharomyces cerevisiae contains more than 1000 proteins, majority of which are imported from the cytosol. The TIM23 and the TIM22 complexes in the inner mitochondrial membrane are essential transport systems for proper insertion of inner membrane and matrix proteins in mitochondria. Additionally, both complexes recognise different targeting signals on a precursor protein. In this study, the TIM23 and the TIM22 complexes were investigated regarding their structure and substrate spectrum respectively. The TOM and the TIM23 complexes cooperate for importing presequence-containing proteins into mitochondria. However, the lack of structural information of the TIM23 complex prevents us from completely deciphering the exact mechanism for the import of a presequence-containing substrate. In this study, we designed and generated new proteins for formation of the TOM-TIM23 supercomplex in organello and in vivo. Subsequently, we optimised the isolation strategy to obtain preparatory amounts of the supercomplex for structural and cross-linking analysis. Through our cross-linking analysis, we mapped the interaction between subunits of the TIM23 complex in its unoccupied state and its translocation intermediate TOM-TIM23 supercomplex state. We identified cross-links between Tim23-Tom40, Tim21-Mgr2 and Hsp70-Mge1 in the unoccupied TIM23 complex. Additionally, cross-links between Tom22-Tim21, Pam16-Tim44 and Pam16-Pam18 were also identified in the supercomplex state of TIM23. Together, these suggest the dynamic nature of interactions within the subunits of the PAM complex, as well as between TOM and TIM23 subunits, during the process of translocation of a protein into mitochondria. The TIM22 complex is required for the import of polytopic inner membrane proteins which lack a presequence but have internal targeting signals. These proteins have predominantly been defined to contain either four or six transmembrane domains. However, so far, only a few proteins have been identified as being substrates of this complex. Therefore, the primary aim of the second project was to expand the substrate spectrum of the TIM22 complex. For this, we utilised a Tim22 temperature sensitive strain in combination with quantitative mass spectrometry. Numerous proteins belonging to the carrier family, such as Crc1, Odc1, Yhm2 and Hem25, were confirmed as substrates of the TIM22 complex. Moreover, previously uncharacterised proteins YPR011C and YFR045W were also identified as TIM22 substrates. Together, these results expand our knowledge about the molecular interactions between mitochondrial translocase components during active protein import, as well as increase our repertoire of the TIM22 complex substrates.
Keywords: Mitochondria; Import; presequence pathway; carrier pathway
 
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