Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation.

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 ß-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 ß-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring ß-barrel formation in vivo and in organello and demonstrated that the ß-barrel was formed and membrane inserted while the precursor was bound to SAM. ß-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.

Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane.

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.

In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins.

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40-substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40-substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.

Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis.

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.

Identification of the signal directing Tim9 and Tim10 into the intermembrane space of mitochondria.

The intermembrane space of mitochondria contains the specific mitochondrial intermembrane space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of intermembrane space proteins. The elements that are crucial for the association of the intermembrane space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the intermembrane space. Thus, here we have identified the mitochondrial intermembrane space sorting signal required for delivery of proteins to the mitochondrial intermembrane space.

Mitochondrial biogenesis, switching the sorting pathway of the intermembrane space receptor Mia40.

Mitochondrial precursor proteins are directed into the intermembrane space via two different routes, the presequence pathway and the redox-dependent MIA pathway. The pathways were assumed to be independent and transport different proteins. We report that the intermembrane space receptor Mia40 can switch between both pathways. In fungi, Mia40 is synthesized as large protein with an N-terminal presequence, whereas in metazoans and plants, Mia40 consists only of the conserved C-terminal domain. Human MIA40 and the C-terminal domain of yeast Mia40 (termed Mia40(core)) rescued the viability of Mia40-deficient yeast independently of the presence of a presequence. Purified Mia40(core) was imported into mitochondria via the MIA pathway. With cells expressing both full-length Mia40 and Mia40(core), we demonstrate that yeast Mia40 contains dual targeting information, directing the large precursor onto the presequence pathway and the smaller Mia40(core) onto the MIA pathway, raising interesting implications for the evolution of mitochondrial protein sorting.

Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase.

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.

Biogenesis of the essential Tim9-Tim10 chaperone complex of mitochondria: site-specific recognition of cysteine residues by the intermembrane space receptor Mia40.

The mitochondrial intermembrane space (IMS) contains an essential machinery for protein import and assembly (MIA). Biogenesis of IMS proteins involves a disulfide relay between precursor proteins, the cysteine-rich IMS protein Mia40 and the sulfhydryl oxidase Erv1. How precursor proteins are specifically directed to the IMS has remained unknown. Here we systematically analyzed the role of cysteine residues in the biogenesis of the essential IMS chaperone complex Tim9-Tim10. Although each of the four cysteines of Tim9, as well as of Tim10, is required for assembly of the chaperone complex, only the most amino-terminal cysteine residue of each precursor is critical for translocation across the outer membrane and interaction with Mia40. Mia40 selectively recognizes cysteine-containing IMS proteins in a site-specific manner in organello and in vitro. Our results indicate that Mia40 acts as a trans receptor in the biogenesis of mitochondrial IMS proteins.

Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex.

Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Little is known about the functional cooperation of these translocases. We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS). The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex. Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex. Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex. Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.

L23 protein functions as a chaperone docking site on the ribosome.

During translation, the first encounter of nascent polypeptides is with the ribosome-associated chaperones that assist the folding process--a principle that seems to be conserved in evolution. In Escherichia coli, the ribosome-bound Trigger Factor chaperones the folding of cytosolic proteins by interacting with nascent polypeptides. Here we identify a ribosome-binding motif in the amino-terminal domain of Trigger Factor. We also show the formation of crosslinked products between Trigger Factor and two adjacent ribosomal proteins, L23 and L29, which are located at the exit of the peptide tunnel in the ribosome. L23 is essential for the growth of E. coli and the association of Trigger Factor with the ribosome, whereas L29 is dispensable in both processes. Mutation of an exposed glutamate in L23 prevents Trigger Factor from interacting with ribosomes and nascent chains, and causes protein aggregation and conditional lethality in cells that lack the protein repair function of the DnaK chaperone. Purified L23 also interacts specifically with Trigger Factor in vitro. We conclude that essential L23 provides a chaperone docking site on ribosomes that directly links protein biosynthesis with chaperone-assisted protein folding.

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins.

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.