Mitochondrial Signalling and protein turnover
Mitochondria are central organelles in eukaryotic cells. Beside their important role in the production of cellular ATP by OXPHOS as well as many biosynthesis pathways, mitochondria represent the central switchpoint in the induction of programmed cell death. More than 98% of all mitochondrial proteins are nuclear encoded and have to be imported into the organelle after their synthesis as precursors in the cytosol. Several sophisticated transport machineries mediate recognition, transport, sorting and assembly of these precursors. The yeast Saccharomyces cerevisiae is an ideal modell organism to study such procecsses. It can be easily genetically manipulated and isolated mitochondria are quite stable which is crucial for e.g. in organello experiments.
Our main research interests are the analysis of the protein composition of mitochondria and their subcompartments and how proteins are sorted to these compartments during biogenesis. Moreover, recent discoveries from our lab revealed unexpected novel mechanisms that regulate protein import and assembly by posttranslational modifications and connect precursor presequence processing to protein turnover.
The proteome of mitochondria and the identification of novel protein import pathways
In collaboration with the groups of A. Sickmann (ISAS, Dortmund) and H. Meyer (Proteome Center, Bochum) we could uncover the proteome of mitochondria from the yeast Saccharomyces cerevisiae with a coverage of almost 90%. We identified 851 different proteins with approx. 250 proteins of so far unknown function (Sickmann et al., PNAS 2003; Reinders et al., J. Proteome Res. 2006; Meisinger et al., Cell 2008).
Fig. 1 Functional classification of the mitochondrial proteome of yeast.
Within this pool of novel mitochondrial proteins we could discover novel protein import and assembly systems in mitochondria (Schmidt et al., Nat. Rev. Mol. Cell Biol. 2010) including the novel MIA import pathway for intermembrane space proteins (Chacinska et al. 2004 EMBO J.;
Gabriel et al., J. Mol. Biol. 2007) or Zim17 a heatshock protein, which prevents aggregation of mitochondrial Hsp70 proteins (Sanjuan-Szklarz et al., J. Mol. Biol. 2005). Our proteomics approach also led to the discovery of a novel sorting and assembly machinery (SAM-complex) in the outer membrane including four different components, Sam50, Sam35, Sam37 and Mdm10. Functional analysis revealed a role of the SAM-complex in sorting and assembly of outer membrane proteins with complicated membrane topologies such like b-barrel proteins (Wiedemann et al., Nature 2003; Meisinger et al., Dev Cell 2004; Stojanovski et al., J. Cell Biol. 2007; Meisinger et al., EMBO J. 2007). Some of these novel proteins we found to be homologues of human disease genes which had never been linked to mitochondria. One example here represents Hfd1, a homolog of the human fatty aldehyde dehydrogenase, which causes the neurodegenerative disease Sjögren-Larsson-Syndrome (Zahedi et al., Mol. Biol. Cell 2006).
Fig. 2 Discovery of the SAM-complex as a novel Mitochondrial protein import machinery
Systematic profiling of mitochondria for posttranslational modifications brought us to the first phospho proteome of mitochondria with 80 different phosphorylation sites including a role of phosphorylation for oligomerization of the ATP-synthase (Reinders et al., Mol. Cell. Proteomics 2007). In order to increase the sensitivity for detection of phosphorylation sites we purified mitochondrial outer membrane vesicles which led then to the identification of more than 100 different phospho sites including many components of the mitochondrial protein entry gate TOM (Schmidt et al., Cell 2011; unpublished data).
Signaltransduction in mitochondria
Mitochondria had been considered for a long time as quite autonomous organelles. Mechanisms of signaltransduction between mitochondria and other cellular compartments were studied only marginally. For the most common principle of signal transmission, reversible phosphorylation, only two examples had been established in yeast mitochondria. Our studies revealed the presence of many phosphorylated proteins in mitochondria in vivo as well as 15 different protein kinases and six phosphatases (see above). This enables us to study principles of signaltransduction within and at mitochondria and to define (on a long perspective) a mitochondrial SIGNALOSOME.
The unexpected identification of more than 100 different phosphorylation sites at the mitochondrial outer membrane indicates that this mitochondrial subcompartment reflects a major signal integration platform that coordinates regulatory mechanisms between mitochondria and other cellular compartments (e.g. cytosol).
Fig.3 Phosphorylation of the mitochondrial protein entry gate TOM by cytosolic protein kinases (Schmidt et al., Cell 2011)
We found 30 different phosphorylation sites at proteins of the central mitochondrial protein entry gate TOM (Fig. 3) and could identify first signalling mechanisms that regulate mitochondrial biogenesis by cytosolic protein kinases (Schmidt et al., Cell 2011; Rao et al., Cell Cycle 2011; Rao et al., Mol. Biol Cell 2012): While Casein kinase 2 plays a general role in the maintenance of the TOM-complex, Protein kinase A exerts a metabolic switch that adapts import of mitochondrial proteins to the metabolic state of yeast cells (Fig. 4).
Fig. 4 Regulation of mitochondrial protein import by the cytosolic kinases CK2 and PKA.
Signaltransduction within and at mitochondria will be a major focus of my lab in the future. We will combine systematic, proteomic and functional analysis to uncover novel signalling systems that regulate mitochondrial functions, e.g. dependent on various cell cycle states or during stress responses. We discovered mitochondrial import proteins as substrates of cyclin dependent kinases and MAP kinases and will analyze this novel link between cell cycle regulation and mitochondrial biogenesis. We will also study the role of intra-mitochondrial protein kinases and phosphatases which have been found by our proteomic studies.
Mitochondrial protein turnover: Discovery of an organellar N-end rule
Approximately 70% of mitochondrial preproteins contain N-terminal presequences that are cleaved by the matrix presequence peptidase MPP after import into the organelle via the presequence pathway. Sorting into further mitochondrial subcompartments can be achieved by various processing peptidases that have been identified (Fig. 5). By using the COFRADIC proteomic approach (in collaboration with K. Gevaert, Ghent and A. Sickmann, Dortmund) we systematically determined the mature N-termini of > 600 mitochondrial proteins. The resulting N-proteome serves as a highly valuable tool to identify novel mitochondrial signal sequences and import mechanisms (Vögtle et al., 2009).
Fig. 5 Overview of presequence processing and degradation enzymes in mitochondria.
For example, for a subclass of those N-termini we surprisingly found that they are processed by a novel peptidase (Icp55) which cleaves off single amino acid residues at the N-terminus thereby converting destabilizing N-termini into stabilizing ones. This led to the discovery of the existence of a mitochondrial N-end rule. We could also show this novel principle with the matrix octapeptidyl peptidase Oct1 which removes an octapetide after intial processing by MPP (Fig. 6; Vögtle et al., 2011).