Ammy C. Maarse

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70–Tim44

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70–Tim44 driving system. By blue native electrophoresis, we identify an ∼90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence‐containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino‐terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy‐terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.

Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane

A screening for yeast mutants impaired in mitochondrial protein import led to the identification of two genes (MPI1 and MPI2) encoding the essential components MIM44 and MIM17 of the inner membrane import machinery. We analyzed twelve additional mutants obtained in the screening and found two further complementation groups. One group represents mutants of SSC1, the gene encoding mitochondrial hsp70, an essential matrix protein required for protein import across the inner membrane. The second complementation group represents mutants of a new gene (MPI3) encoding a 23 kDa integral inner membrane protein (MIM23). MIM23 is synthesized without a presequence, and its import to the inner membrane requires a membrane potential. MIM23 contains a domain homologous to half of MIM17. We speculate that MIM23 is a new member of the protein import machinery of the mitochondrial inner membrane.

Identification of the essential yeast protein MIM17, an integral mitochondrial inner membrane protein involved in protein import

We analyzed four Saccharomyces cerevisiae mutants defective in mitochondrial protein import and found that they are complemented by a novel gene encoding a 17 kDa protein. The protein is integrally located in the mitochondrial inner membrane and is termed MIM17. It shows significant homology to MIM23/Mas6p, a previously identified mitochondrial inner membrane protein required for the import of preproteins. Like MIM23, the precursor of MIM17 is synthesized without a presequence. A deletion of MIM17 is lethal. MIM17 thus joins the small group of mitochondrial proteins that are essential for the viability of yeast. We propose that MIM17 is an essential component of the preprotein import machinery of the mitochondrial inner membrane.

Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein.

The six small subunits (IV‐VII, VIIa, VIII) of yeast cytochrome c oxidase are encoded by nuclear genes and imported into the mitochondria. We have isolated the gene for subunit IV from a yeast genomic clone bank and determined its complete nucleotide sequence. We have also isolated subunit IV from purified yeast cytochrome c oxidase and determined most of its amino acid sequence which confirms the positioning of approximately 90% of the amino acid residues. The sequence comparison shows that the coding sequence of the gene lacks introns and that subunit IV is made as a precursor with an amino‐terminal extension of 25 residues, five of which are basic and none of them acidic. Precursor processing involves cleavage of a Leu‐Gln bond.

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat‐shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by ∼30% compared with wild‐type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.

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.

Isolation, characterization and regulation of expression of the nuclear genes for the core II and Rieske iron-sulphur proteins of the yeast ubiquinol-cytochrome c reductase

Cloning and mapping of the yeast nuclear genes for the core II (Mr 40 000) and Rieske iron-sulphur proteins of the mitochondrial ubiquinol-cytochrome c reductase, and comparison with the genomic regions in nuclear DNA from which they are derived, show that the genes are likely to be present in single copies and that they are not closely linked. They have been reintroduced into yeast cells on multi-copy plasmids and, similar to results obtained for the Mr 11 000 subunit [Van Loon et al., EMBO J. 2 (1983) 1765-1770], increase in the dosage of either gene prompts discoordinate synthesis of the encoded protein. Quantitative analysis of transformants carrying extra copies of the gene for the iron-sulphur protein shows that messenger RNA level, rate of synthesis and steady-state concentration of the protein correlate well with each other. This indicates that its level, in contrast to that of the Mr 11 000 subunit, is only determined by the concentration of its messenger RNA. Over-production of these proteins does not interfere with mitochondrial function as judged from growth rates of transformed cells on non-fermentable media. The excess Mr 40 000 protein is imported into the mitochondrion, showing that import of this subunit is not obligatorily coupled to complex assembly.

Epitope-tagging vectors designed for yeast.

In order to facilitate the process of epitope-tagging of yeast proteins, we have constructed two Saccharomyces cerevisiae-Escherichia coli shuttle vectors that allow fusion of a sequence encoding an epitope of the human c-myc protein at the 3′ end of any gene. An example of the use of this technique is presented.