A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.

Intermembrane space proteome of yeast mitochondria.

The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.

PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy.

Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1's and PLK1's functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.

Mitochondrial protein import receptors in Kinetoplastids reveal convergent evolution over large phylogenetic distances.

Mitochondrial protein import is essential for all eukaryotes and mediated by hetero-oligomeric protein translocases thought to be conserved within all eukaryotes. We have identified and analysed the function and architecture of the non-conventional outer membrane (OM) protein translocase in the early diverging eukaryote Trypanosoma brucei. It consists of six subunits that show no obvious homology to translocase components of other species. Two subunits are import receptors that have a unique topology and unique protein domains and thus evolved independently of the prototype receptors Tom20 and Tom70. Our study suggests that protein import receptors were recruited to the core of the OM translocase after the divergence of the major eukaryotic supergroups. Moreover, it links the evolutionary history of mitochondrial protein import receptors to the origin of the eukaryotic supergroups.

Mitochondria. Cell cycle-dependent regulation of mitochondrial preprotein translocase.

Mitochondria play central roles in cellular energy conversion, metabolism, and apoptosis. Mitochondria import more than 1000 different proteins from the cytosol. It is unknown if the mitochondrial protein import machinery is connected to the cell division cycle. We found that the cyclin-dependent kinase Cdk1 stimulated assembly of the main mitochondrial entry gate, the translocase of the outer membrane (TOM), in mitosis. The molecular mechanism involved phosphorylation of the cytosolic precursor of Tom6 by cyclin Clb3-activated Cdk1, leading to enhanced import of Tom6 into mitochondria. Tom6 phosphorylation promoted assembly of the protein import channel Tom40 and import of fusion proteins, thus stimulating the respiratory activity of mitochondria in mitosis. Tom6 phosphorylation provides a direct means for regulating mitochondrial biogenesis and activity in a cell cycle-specific manner.

From inventory to functional mechanisms: regulation of the mitochondrial protein import machinery by phosphorylation.

For decades, the pyruvate dehydrogenase complex in the mitochondrial matrix was considered as a rare example of how protein kinases and phosphatases can regulate important functions within this organelle. During the last decade, several proteomic studies revealed that a large fraction of mitochondrial proteins are indeed phosphorylated. A surprisingly high number of phosphorylation sites was found at the preprotein import machinery, TOM, in the outer membrane that provides the central protein import gate for most mitochondrial precursors synthesized in the cytosol. This review describes current knowledge of the mitochondrial phosphoproteome and introduces the first regulatory mechanisms of protein import dynamics by reversible phosphorylation, which have been uncovered mainly in the model organism Saccharomyces cerevisiae.

Glucose-induced regulation of protein import receptor Tom22 by cytosolic and mitochondria-bound kinases.

Most mitochondrial proteins are imported by the translocase of the outer mitochondrial membrane (TOM). Tom22 functions as central receptor and transfers preproteins to the import pore. Casein kinase 2 (CK2) constitutively phosphorylates the cytosolic precursor of Tom22 at Ser44 and Ser46 and, thus, promotes its import. It is unknown whether Tom22 is regulated under different metabolic conditions. We report that CK1, which is involved in glucose-induced signal transduction, is bound to mitochondria. CK1 phosphorylates Tom22 at Thr57 and stimulates the assembly of Tom22 and Tom20. In contrast, protein kinase A (PKA), which is also activated by the addition of glucose, phosphorylates the precursor of Tom22 at Thr76 and impairs its import. Thus, PKA functions in an opposite manner to CK1 and CK2. Our results reveal that three kinases regulate the import and assembly of Tom22, demonstrating that the central receptor is a major target for the posttranslational regulation of mitochondrial protein import.

Signaling at the gate: phosphorylation of the mitochondrial protein import machinery.

Protein import into mitochondria is an essential process in every eukaryotic organism. While most of the components of the import machinery have been identified and are mechanistically quite well understood, regulation of this process had been a largely neglected area of research in the past. Recently, we demonstrated for the first time that the translocase of the outer mitochondrial membrane (TOM) is phosphorylated and regulated by several cytosolic protein kinases. Among these, casein kinase 2 (CK2) governs the assembly of TOM complexes, while protein kinase A (PKA) controls translocase function. Here, we outline the current model of protein import regulation, together with additional mitochondrial functions of CK2 and PKA. We also reflect the data on mitochondria-associated protein kinases and phosphatases in the model organism baker's yeast.