RESUMO
A major challenge in eukaryotic cells is the proper distribution of nuclear-encoded proteins to the correct organelles. For a subset of mitochondrial proteins, a signal sequence at the N terminus (matrix-targeting sequence [MTS]) is recognized by protein complexes to ensure their proper translocation into the organelle. However, the early steps of mitochondrial protein targeting remain undeciphered. The cytosolic chaperone nascent polypeptide-associated complex (NAC), which in yeast is represented as the two different heterodimers αß-NAC and αß'-NAC, has been proposed to be involved during the early steps of mitochondrial protein targeting. We have previously described that the mitochondrial outer membrane protein Sam37 interacts with αß'-NAC and together promote the import of specific mitochondrial precursor proteins. In this work, we aimed to detect the region in the MTS of mitochondrial precursors relevant for their recognition by αß'-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αß'-NAC-dependent Oxa1 and αß'-NAC-independent Mdm38) and fused them to GFP to study their intracellular localization by biochemical and microscopy methods, and in addition followed their import kinetics in vivo. Our results reveal the presence of a positively charged amino acid cluster in the MTS of select mitochondrial precursors, such as Oxa1 and Fum1, which are crucial for their recognition by αß'-NAC. Furthermore, we explored the presence of this cluster at the N terminus of the mitochondrial proteome and propose a set of precursors whose proper localization depends on both αß'-NAC and Sam37.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Mitocondriais , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Cancer is characterized by an uncontrolled cell proliferation rate even under low nutrient availability, which is sustained by a metabolic reprograming now recognized as a hallmark of cancer. Warburg was the first to establish the relationship between cancer and mitochondria; however, he interpreted enhanced aerobic glycolysis as mitochondrial dysfunction. Today it is accepted that many cancer cell types need fully functional mitochondria to maintain their homeostasis. Calcium (Ca2+)-a key regulator of several cellular processes-has proven to be essential for mitochondrial metabolism. Inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ transfer from the endoplasmic reticulum to the mitochondria through the mitochondrial calcium uniporter (MCU) proves to be essential for the maintenance of mitochondrial function and cellular energy balance. Both IP3R and MCU are overexpressed in several cancer cell types, and the inhibition of the Ca2+ communication between these two organelles causes proliferation arrest, migration decrease, and cell death through mechanisms that are not fully understood. In this review, we summarize and analyze the current findings in this area, emphasizing the critical role of Ca2+ and mitochondrial metabolism in cancer and its potential as a novel therapeutic target.
RESUMO
To accomplish its crucial role, mitochondria require proteins that are produced in the cytosol, delivered by cytosolic Hsp90, and translocated to its interior by the translocase outer membrane (TOM) complex. Hsp90 is a dimeric molecular chaperone and its function is modulated by its interaction with a large variety of co-chaperones expressed within the cell. An important family of co-chaperones is characterized by the presence of one TPR (tetratricopeptide repeat) domain, which binds to the C-terminal MEEVD motif of Hsp90. These include Tom70, an important component of the TOM complex. Despite a wealth of studies conducted on the relevance of Tom70·Hsp90 complex formation, there is a dearth of information regarding the exact molecular mode of interaction. To help fill this void, we have employed a combined experimental strategy consisting of cross-linking/mass spectrometry to investigate binding of the C-terminal Hsp90 domain to the cytosolic domain of Tom70. This approach has identified a novel region of contact between C-Hsp90 and Tom70, a finding that is confirmed by probing the corresponding peptides derived from cross-linking experiments via isothermal titration calorimetry and mitochondrial import assays. The data generated in this study are combined to input constraints for a molecular model of the Hsp90/Tom70 interaction, which has been validated by small angle x-ray scattering, hydrogen/deuterium exchange, and mass spectrometry. The resultant model suggests that only one of the MEEVD motifs within dimeric Hsp90 contacts Tom70. Collectively, our findings provide significant insight on the mechanisms by which preproteins interact with Hsp90 and are translocated via Tom70 to the mitochondria.