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.

Biogenesis of mitochondrial β-barrel proteins requires two preprotein translocases, the general translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). TOM and SAM form a supercomplex that promotes transfer of β-barrel precursors. The SAM core complex contains the channel protein Sam50, which cooperates with Sam35 in precursor recognition, and the peripheral membrane protein Sam37. The molecular function of Sam37 has been unknown. We report that Sam37 is crucial for formation of the TOM–SAM supercomplex. Sam37 interacts with the receptor domain of Tom22 on the cytosolic side of the mitochondrial outer membrane and links TOM and SAM complexes. Sam37 thus promotes efficient transfer of β-barrel precursors to the SAM complex. We conclude that Sam37 functions as a coupling factor of the translocase supercomplex of the mitochondrial outer membrane.

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.

Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain. We have identified a new subunit of the TIM23 complex, the inner membrane protein Mgr2. Mitochondria lacking Mgr2 were deficient in the Tim21-containing sorting form of the TIM23 complex. Mgr2 was required for binding of Tim21 to TIM23 CORE , revealing a binding chain of TIM23 CORE -Mgr2/Tim21–respiratory chain. Mgr2-deficient yeast cells were defective in growth at elevated temperature, and the mitochondria were impaired in TOM-TIM23 coupling and the import of presequence-carrying preproteins. We conclude that Mgr2 is a coupling factor of the presequence translocase crucial for cell growth at elevated temperature and for efficient protein import.

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.

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.

The mitochondrial presequence translocase transports preproteins to either matrix or inner membrane. Two different translocase forms have been identified: the matrix transport form, which binds the heat-shock protein 70 (Hsp70) motor, and the inner membrane–sorting form, which lacks the motor but contains translocase of inner mitochondrial membrane 21 (Tim21). The sorting form interacts with the respiratory chain in a Tim21-dependent manner. It is unknown whether the respiratory chain–bound translocase transports preproteins and how the switch between sorting form and motor form occurs. We report that the respiratory chain–bound translocase contains preproteins in transit and, surprisingly, not only sorted but also matrix-targeted preproteins. Presequence translocase-associated motor (Pam) 16 and 18, two regulatory components of the six-subunit motor, interact with the respiratory chain independently of Tim21. Thus, the respiratory chain–bound presequence translocase is not only active in preprotein sorting to the inner membrane but also in an early stage of matrix translocation. The motor does not assemble en bloc with the translocase but apparently in a step-wise manner with the Pam16/18 module before the Hsp70 core.

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the β-barrel–specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a β-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane α-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of α-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to β-barrel proteins but also includes the majority of α-helical Tom proteins.

Most mitochondrial proteins are synthesized in the cytosol and imported into one of the four mitochondrial compartments: outer membrane, intermembrane space, inner membrane, and matrix. Each compartment contains protein complexes that interact with precursor proteins and promote their transport. These translocase complexes do not act as independent units but cooperate with each other and further membrane complexes in a dynamic manner. We propose that a regulated coupling of translocases is important for the coordination of preprotein translocation and efficient sorting to intramitochondrial compartments.

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase–associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18 /YLR008c/ TIM14 ) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro–imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44 Δ18 in addition to the endogenous wild-type Tim44. Tim44 Δ18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44 Δ18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44 Δ18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.