Angelika Hönlinger

Tom5 functionally links mitochondrial preprotein receptors to the general import pore

Most mitochondrial proteins are synthesized as preproteins on cytosolic polysomes and are subsequently imported into the organelle. The mitochondrial outer membrane contains a multisubunit preprotein translocase (Tom) which has receptors on the cytosolic side and a general import pore (GIP) in the membrane. Tom20–Tom22 and Tom70–Tom37 function as import receptors with a preference for preproteins that have amino-terminal presequences or internal targeting information, respectively. Tom40 is an essential constituent of the GIP,, whereas Tom6 and Tom7 modulate the assembly and dissociation of the Tom machinery,. Here we report the identification of Tom5, a small subunit that has a crucial role importing preproteins destined for all four mitochondrial subcompartments. Tom5 has a single membrane anchor and a cytosolic segment with a negative net charge, and accepts preproteins from the receptors and mediates their insertion into the GIP. We conclude that Tom5 represents a functional link between surface receptors and GIP, and is part of an ‘acid chain’ that guides the stepwise transport of positively charged mitochondrial targeting sequences.

Preprotein Translocase of the Outer Mitochondrial Membrane: Molecular Dissection and Assembly of the General Import Pore Complex

ABSTRACT The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of ∼400,000 (∼400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of ∼100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.

TOM7 MODULATES THE DYNAMICS OF THE MITOCHONDRIAL OUTER MEMBRANE TRANSLOCASE AND PLAYS A PATHWAY-RELATED ROLE IN PROTEIN IMPORT

The preprotein translocase of the outer mitochondrial membrane is a multi‐subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re‐establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.

The Mitochondrial Receptor Complex: Mom22 Is Essential for Cell Viability and Directly Interacts with Preproteins†

A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.

The Mitochondrial Receptor Complex: the Small Subunit Mom8b/Isp6 Supports Association of Receptors with the General Insertion Pore and Transfer of Preproteins

The mitochondrial outer membrane contains import receptors for preproteins and a multisubunit general insertion pore. Several small outer membrane proteins (< 10 kDa) have been identified by their association with receptors or the general insertion pore, yet little is known about their function. Here, we present evidence that the biochemically identified Mom8b and the genetically identified Isp6 are identical. A deletion of Mom8b/Isp6 in Saccharomyces cerevisiae leads to (i) a delay of import of preproteins, (ii) stabilization of preprotein binding to receptors and the general insertion pore, and (iii) destabilization of the interaction between receptors and the general insertion pore. These results suggest that Mom8b supports the cooperativity between receptors and the general insertion pore and facilitates the release of preproteins from import components and thereby promotes efficient transfer of preproteins.

The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences.

Mitochondrial protein import is thought to involve the sequential interaction of preproteins with binding sites on cis and trans sides of the membranes. For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) and then are translocated through a general import pore, in a process proposed to involve binding to a trans site on the intermembrane space (IMS) side. Controversial results have been reported for the role of the IMS domain of the essential outer membrane protein Tom22 in formation of the trans site. We show with different mutant mitochondria that a lack of the IMS domain only moderately reduces the direct import of preproteins with N-terminal targeting sequences. The dependence of import on the IMS domain of Tom22 is significantly enhanced by removing the cytosolic domains of import receptors or by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in the absence of a membrane potential (delta psi) and subsequent import after reestablishment of a delta psi. After the removal of cytosolic receptor domains, two-step import of a cleavable preprotein strictly requires the IMS domain. In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22. We conclude that the negatively charged IMS domain of Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences, in agreement with the acid chain hypothesis of mitochondrial protein import.

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source

The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate–phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild‐type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The ΔDIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.

Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane

The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins. Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP‐driven import motor. For this report we characterized the biogenesis and interactions of Tim proteins. (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44. (iii) Depletion of Tim44 does not affect the co‐precipitation of Tim17 with antibodies directed against mtHsp70. (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44–Tim23‐containing sub‐complex and a Tim23–Tim17‐containing sub‐complex. (v) The association of mtHsp70 with the Tim23–Tim17 sub‐complex is ATP sensitive and can be distinguished from the mtHsp70–Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70. (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub‐complexes, each of which transiently binds mtHsp70.

The Import Route of ADP/ATP Carrier into Mitochondria Separates from the General Import Pathway of Cleavable Preproteins at thetrans Side of the Outer Membrane

The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.

[19] Mitochondrial receptor complex from Neurospora crassa and Saccharomyces cerevisiae

Publisher Summary The identification of the mitochondrial receptor complex in yeast and N. crassa may be viewed as a paradigm of how biochemical approaches aimed at the study of protein–protein interactions can be successfully used for characterizing the different components of a protein complex, once the first component has been identified. Structural studies of the mitochondrial receptor complex will provide an enormous wealth of information on the molecular details involved in the interaction among the different components of the complex and between these components and the precursor proteins. The development of optical biosensor- based methodologies for studying protein–protein interactions will provide additional tools for this kind of molecular analysis. As the ultimate goal, the reconstitution of the fully functional translocation machinery into artificial lipid vesicles from purified complex components should provide detailed mechanistic information about the process of the translocation of precursor proteins into mitochondria.