The mitochondrial unfolded protein response in Saccharomyces cerevisiae

• Applicant: Dr. Nikola Wagener
• Subject Area: Cell Biology
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 290153195

Mitochondria are under the control of two genetic systems, the nuclear and the mitochondrial genome. Therefore, mitochondrial homeostasis requires an elaborate crosstalk between both genomes. Regulatory mechanisms depend on various factors, such as the availability of metabolites, oxidative stress, the integrity of membrane potential or protein folding stress. Only a few of these factors have been studied so far. The response to the accumulation of unfolded or incorrectly folded proteins is of particular relevance. Such kind of stress can arise from (a) unbalanced expression of proteins that must assemble into complexes, (b) synthesis and import of altered proteins, (c) damage of mitochondrial DNA, or (d) alterations in transcription/translation. So far, responses to protein folding stress have been observed in Caenorhabditis elegans and mammalian cells; however, our molecular understanding of these responses is still very limited. Due to its excellent suitability for genetic manipulation and biochemical analyses, I propose to use the yeast Saccharomyces cerevisiae as a model to comprehensively investigate the unfolded protein response of mitochondria (UPRmt). Our recent results demonstrate that inhibition of mitochondrial protein synthesis results in increased expression of a set of mitochondrial proteins, in particular Hsp60, mtHsp70, Yme1, Mdj1 and the poorly characterized AAA protein, Msp1. Msp1 mutants show altered levels of small mitochondrial intermembrane space proteins such as Cox17, Tim10 and Tim13. Based on our previous and current work, I propose that Msp1 plays a role in import or export and the subsequent degradation of these intermembrane space proteins. During the funding period, we will identify additional S. cerevisiae proteins (within and outside of mitochondria) that are involved in UPRmt. These proteins will comprise target proteins that exhibit altered expression levels in response to the induction of UPRmt. They will also comprise signaling factors that change localization upon UPRmt induction or are post-translationally modified. This will be achieved by mass spectrometry of mitochondria isolated from normal and stressed cells (SILAC). In addition, proteomes of whole cells and isolated nuclei will be generated. Furthermore, RNAseq analyses will be performed to complement these analyses at the transcriptional level. Another approach to identify proteins involved in UPRmt will be to screen a yeast deletion library using a UPRmt reporter system, which we will develop. This genetic analysis will also extend and validate the SILAC data. Finally, deletion and over-expression mutants as well as tagged variants of identified proteins will be analyzed regarding their growth phenotype, stress sensitivity, mitochondrial structure and interaction partners.

• DFG Programme: Research Grants