Birgit Schönfisch

The proteome of Saccharomyces cerevisiae mitochondria

We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent ≈90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.

Machinery for protein sorting and assembly in the mitochondrial outer membrane

Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes. It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11–15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.

Regulation of Mitochondrial Protein Import by Cytosolic Kinases

Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria.

Mitochondrial Protein Sorting DIFFERENTIATION OF β-BARREL ASSEMBLY BY Tom7-MEDIATED SEGREGATION OF Mdm10

The mitochondrial outer membrane contains two distinct machineries for protein import and protein sorting that function in a sequential manner: the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex), which is dedicated to β-barrel proteins. The SAMcore complex consists of three subunits, Sam35, Sam37, and Sam50, that can associate with a fourth subunit, the morphology component Mdm10, to form the SAMholo complex. Whereas the SAMcore complex is required for the biogenesis of all β-barrel proteins, Mdm10 and the SAMholo complex play a selective role in β-barrel biogenesis by promoting assembly of Tom40 but not of porin. We report that Tom7, a conserved subunit of the TOM complex, functions in an antagonistic manner to Mdm10 in biogenesis of Tom40 and porin. We show that Tom7 promotes segregation of Mdm10 from the SAMholo complex into a low molecular mass form. Upon deletion of Tom7, the fraction of Mdm10 in the SAMholo complex is significantly increased, explaining the opposing functions of Tom7 and Mdm10 in β-barrel sorting. Thus the role of Tom7 is not limited to the TOM complex. Tom7 functions in mitochondrial protein biogenesis by a new mechanism, segregation of a sorting component, leading to a differentiation of β-barrel assembly.

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat‐shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by ∼30% compared with wild‐type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.

Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import

The translocase of the outer mitochondrial membrane (TOM) is essential for the import of proteins into mitochondria. Cytosolic protein kinase A phosphorylates the precursor of the channel-forming protein Tom40 and inhibits its import into mitochondria, thus regulating the biogenesis of the protein entry gate of 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. Cell division and mitochondrial protein import are directly linked by cyclin-dependent phosphorylation of a mitochondrial assembly factor. [Also see Perspective by Schulz and Rehling] Cross talk between mitochondria and mitosis Mitochondria, the power houses of the cell, contain their own genome. Nevertheless, the majority of their constituent proteins are encoded by nuclear genes and are translated in the cytosol. During the cell cycle, a cell needs to duplicate each of its constituent parts and organelles. Surprisingly, it has never been clear how or whether mitochondrial protein import is linked with the cell cycle. Harbauer et al. (see the Perspective by Schulz and Rehling) now show that one of the main conduits for mitochondrial protein import is directly regulated by phosphorylation during mitosis, and that this in turn promotes respiratory activity. Science, this issue p. 1109