Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, ...thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.
The biogenesis of mitochondria requires the import of a large number of proteins from the cytosol 1, 2. Although numerous studies have defined the proteinaceous machineries that mediate mitochondrial ...protein sorting, little is known about the role of lipids in mitochondrial protein import. Cardiolipin, the signature phospholipid of the mitochondrial inner membrane 3–5, affects the stability of many inner-membrane protein complexes 6–12. Perturbation of cardiolipin metabolism leads to the X-linked cardioskeletal myopathy Barth syndrome 13–18. We report that cardiolipin affects the preprotein translocases of the mitochondrial outer membrane. Cardiolipin mutants genetically interact with mutants of outer-membrane translocases. Mitochondria from cardiolipin yeast mutants, as well as Barth syndrome patients, are impaired in the biogenesis of outer-membrane proteins. Our findings reveal a new role for cardiolipin in protein sorting at the mitochondrial outer membrane and bear implications for the pathogenesis of Barth syndrome.
The preprotein translocase of the outer mitochondrial membrane (TOM) functions as the main entry gate for the import of nuclear-encoded proteins into mitochondria. The major subunits of the TOM ...complex are the three receptors Tom20, Tom22, and Tom70 and the central channel-forming protein Tom40. Cytosolic kinases have been shown to regulate the biogenesis and activity of the Tom receptors. Casein kinase 2 stimulates the biogenesis of Tom22 and Tom20, whereas protein kinase A (PKA) impairs the receptor function of Tom70. Here we report that PKA exerts an inhibitory effect on the biogenesis of the β-barrel protein Tom40. Tom40 is synthesized as precursor on cytosolic ribosomes and subsequently imported into mitochondria. We show that PKA phosphorylates the precursor of Tom40. The phosphorylated Tom40 precursor is impaired in import into mitochondria, whereas the nonphosphorylated precursor is efficiently imported. We conclude that PKA plays a dual role in the regulation of the TOM complex. Phosphorylation by PKA not only impairs the receptor activity of Tom70, but it also inhibits the biogenesis of the channel protein Tom40.
The general preprotein translocase of the outer mitochondrial membrane (TOM complex) transports virtually all mitochondrial precursor proteins, but cannot assemble outer-membrane precursors into ...functional complexes. A recently discovered sorting and assembly machinery (SAM complex) is essential for integration and assembly of outer-membrane proteins, revealing unexpected connections to mitochondrial evolution and morphology.
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.
Biogenesis of the translocase of the outer mitochondrial membrane (TOM complex) involves the assembly of the central β-barrel forming protein Tom40 with six different subunits that are embedded in ...the membrane via α-helical transmembrane segments. The sorting and assembly machinery (SAM complex) of the outer membrane plays a central role in this process. The SAM complex mediates the membrane integration of β-barrel precursor proteins including Tom40. The small Tom proteins Tom5 and Tom6 associate with the precursor of Tom40 at the SAM complex at an early stage of the assembly process and play a stimulatory role in the formation of the mature TOM complex. A fraction of the SAM components interacts with the outer membrane protein mitochondrial distribution and morphology protein 10 (Mdm10) to form the SAM–Mdm10 machinery; however, different views exist on the function of the SAM–Mdm10 complex. We report here that the third small Tom protein, Tom7, plays an inhibitory role at two distinct steps in the biogenesis of the TOM complex. First, Tom7 plays an antagonistic role to Tom5 and Tom6 at the early stage of Tom40 assembly at the SAM complex. Second, Tom7 interacts with Mdm10 that is not bound to the SAM complex, and thus promotes dissociation of the SAM–Mdm10 complex. Since the SAM–Mdm10 complex is required for the biogenesis of Tom22, Tom7 delays the assembly of Tom22 with Tom40 at a late stage of assembly of the TOM complex. Thus, Tom7 modulates the biogenesis of topologically different proteins, the β-barrel forming protein Tom40 and Tom22 that contains a transmembrane α-helix.
Mitochondria contain ∼1000 different proteins, which are located in four different compartments, outer membrane, inner membrane, intermembrane space and matrix. The vast majority of these proteins ...has to be imported from the cytosol. Therefore, sophisticated molecular machineries have evolved that mediate protein translocation across or insertion into mitochondrial membranes and subsequent assembly into multi-subunit complexes. While the initial entry of virtually all mitochondrial proteins is mediated by the general import pore of the outer membrane, at least four different downstream pathways are dedicated to import and assembly of proteins into a specific compartment.
The mitochondrial inner membrane is a highly protein-rich membrane with central importance for oxidative phosphorylation and metabolite transport 1. A large number of inner-membrane proteins are ...synthesized as preproteins with cleavable presequences 2–9. Opposing mechanisms of preprotein insertion into the membrane have been debated: stop-transfer with arrest in the inner membrane versus conservative sorting via the matrix 3, 8, 10. We dissected the membrane insertion of a multispanning ABC transporter. The N-terminal membrane domain was laterally released from the presequence translocase of the inner membrane (TIM23 complex) by a stop-transfer mechanism, whereas the subsequent domain was imported via the matrix heat-shock protein 70 (mtHsp70) motor and exported by the oxidase assembly (OXA) translocase. These observations lead to an unexpected solution to the controversial debate about mitochondrial preprotein sorting. Stop-transfer and conservative sorting are not mutually exclusive pathways but represent sorting mechanisms that cooperate in the membrane integration of a protein with complex topology. We conclude that the multispanning protein is inserted in a modular manner by the coordinated action of two inner-membrane preprotein translocases.
► Different views of mitochondrial protein sorting combined in a cooperative mechanism ► Conservative sorting and stop-transfer mechanism required for the same preprotein ► Insertion of distinct protein domains from opposite sides of the membrane ► Cooperation of TIM import machinery and OXA export machinery in protein insertion
Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for ...maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport-associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.
The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase ...(TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.
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► Subunit of mitochondrial respiratory chain functions in protein import ► Succinate dehydrogenase subunit 3 (Sdh3) is a subunit of TIM22 protein translocase ► Sdh4 and Tim18 recruit Sdh3 to two different membrane protein complexes ► Membrane module of respiratory complex as evolutionary building block for translocase
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