The endoplasmic reticulum-mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of ...mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.
The Protein Import Machinery of Mitochondria Wiedemann, Nils; Frazier, Ann E.; Pfanner, Nikolaus
The Journal of biological chemistry,
04/2004, Volume:
279, Issue:
15
Journal Article
Most mitochondrial proteins are encoded in the nucleus as precursor proteins and carry N-terminal presequences for import into the organelle. The vast majority of presequences are proteolytically ...removed by the mitochondrial processing peptidase (MPP) localized in the matrix. A subset of precursors with a characteristic amino acid motif is additionally processed by the mitochondrial intermediate peptidase (MIP) octapeptidyl aminopeptidase 1 (Oct1), which removes an octapeptide from the N-terminus of the precursor intermediate. However, the function of this second cleavage step is elusive. In this paper, we report the identification of a novel Oct1 substrate protein with an unusual cleavage motif. Inspection of the Oct1 substrates revealed that the N-termini of the intermediates typically carry a destabilizing amino acid residue according to the N-end rule of protein degradation, whereas mature proteins carry stabilizing N-terminal residues. We compared the stability of intermediate and mature forms of Oct1 substrate proteins in organello and in vivo and found that Oct1 cleavage increases the half-life of its substrate proteins, most likely by removing destabilizing amino acids at the intermediate's N-terminus. Thus Oct1 converts unstable precursor intermediates generated by MPP into stable mature proteins.
Invaginations of the mitochondrial inner membrane, termed cristae, are hubs for oxidative phosphorylation. The mitochondrial contact site and cristae organizing system (MICOS) and the dimeric ...F1Fo-ATP synthase play important roles in controlling cristae architecture. A fraction of the MICOS core subunit Mic10 is found in association with the ATP synthase, yet it is unknown whether this interaction is of relevance for mitochondrial or cellular functions. Here, we established conditions to selectively study the role of Mic10 at the ATP synthase. Mic10 variants impaired in MICOS functions stimulate ATP synthase oligomerization like wild-type Mic10 and promote efficient inner membrane energization, adaptation to non-fermentable carbon sources, and respiratory growth. Mic10's functions in respiratory growth largely depend on Mic10ATPsynthase, not on Mic10MICOS. We conclude that Mic10 plays a dual role as core subunit of MICOS and as partner of the F1Fo-ATP synthase, serving distinct functions in cristae shaping and respiratory adaptation and growth.
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•Dual role of Mic10 of mitochondrial contact site and cristae organizing system (MICOS)•Mic10 binds to mitochondrial ATP synthase and stabilizes higher order assemblies•Oligomerization of Mic10 is required for its function in MICOS, not at ATP synthase•Mic10 binding to ATP synthase supports metabolic adaptation and respiratory growth
Rampelt et al. report that the core component Mic10 of the mitochondrial contact site and cristae organizing system plays a second physiologically important role at the mitochondrial ATP synthase. Mic10 variants that selectively function at the ATP synthase promote efficient metabolic adaptation, respiratory growth, and mitochondrial inner membrane energization.
The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer ...membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.
The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to ...SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway.
Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins.
The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.
The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly ...machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.
Background: It is unknown if phosphatidylethanolamine (PE), the major non-bilayer-forming mitochondrial phospholipid, is involved in the biogenesis of outer membrane proteins.
Results: Depletion of PE impairs import of β-barrel proteins by the outer membrane translocase TOM.
Conclusion: PE is required for full activity but not stability of TOM.
Significance: PE plays a different role in the biogenesis of mitochondrial outer membrane proteins compared with cardiolipin.
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.
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•Regulation of mitochondrial protein translocase by glucose-induced signaling pathways•Central mitochondrial receptor Tom22 is regulated by a network of protein kinases•Protein kinase A inhibits Tom22 biogenesis, whereas casein kinases 1 and 2 stimulate it•Dual localization of casein kinase 1 at the plasma membrane and mitochondria
The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly ...machinery (SAM). The precursors of β-barrel proteins use both translocases—TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the β-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring β-barrel formation in vivo and in organello and demonstrated that the β-barrel was formed and membrane inserted while the precursor was bound to SAM. β-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.
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•Native assay monitors β-barrel folding of imported proteins in intact mitochondria•β-barrel folding and membrane insertion occur at sorting and assembly machinery SAM•Receptor Tom22 connects mitochondrial import and export translocases TOM and SAM•Translocase supercomplex promotes precursor channeling
The import and export translocases TOM and SAM collaborate, forming a supercomplex that promotes the channeling of precursor proteins from one translocase to the other during the assembly of β-barrel proteins in the mitochondrial membrane.
The mitochondrial inner membrane plays central roles in bioenergetics and metabolism and contains several established membrane protein complexes. Here, we report the identification of a mega-complex ...of the inner membrane, termed mitochondrial multifunctional assembly (MIMAS). Its large size of 3 MDa explains why MIMAS has escaped detection in the analysis of mitochondria so far. MIMAS combines proteins of diverse functions from respiratory chain assembly to metabolite transport, dehydrogenases, and lipid biosynthesis but not the large established supercomplexes of the respiratory chain, ATP synthase, or prohibitin scaffold. MIMAS integrity depends on the non-bilayer phospholipid phosphatidylethanolamine, in contrast to respiratory supercomplexes whose stability depends on cardiolipin. Our findings suggest that MIMAS forms a protein-lipid mega-assembly in the mitochondrial inner membrane that integrates respiratory biogenesis and metabolic processes in a multifunctional platform.
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•Identification of a 3 MDa complex of the inner mitochondrial membrane•Mitochondrial multifunctional assembly (MIMAS) combines respiratory assembly and metabolism•MIMAS integrity requires the non-bilayer phospholipid phosphatidylethanolamine•The protein-lipid mega-assembly MIMAS functions as a biogenesis and metabolic platform
Horten et al. have identified a lipid-dependent mega-complex of the mitochondrial inner membrane. The mitochondrial multifunctional assembly (MIMAS) includes proteins for respiratory chain biogenesis, metabolism, and phospholipid biosynthesis. MIMAS reveals a higher-level organization of a protein-rich membrane by integrating diverse functions into a protein-lipid mega-assembly.
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