Import of cytochrome c heme lyase into mitochondria

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane

A mutant of Neurospora crassa deficient in cytochrome c heme lyase activity cannot import cytochrome c into mitochondria

The nuclear cyt-2-1 mutant of Neurospora crassa is characterized by a gross deficiency of cytochrome c (Bertrand, H., and Collins, R. A. (1978) Mol. Gen. Genet. 166, 1-13). The mutant produces mRNA that can be translated into apocytochrome c in vitro. Apocytochrome c is also synthesized in vivo in cyt-2-1, but it is rapidly degraded and thus does not accumulate in the cytosol. Mitochondria from wild-type cells bind apocytochrome c made in vitro from either wild-type or cyt-2-1 mRNA and convert it to holocytochrome c. This conversion depends on the addition of heme by cytochrome c heme lyase and is coupled to translocation of cytochrome c into the intermembrane space. Mitochondria from the cyt-2-1 strain are deficient in the ability to bind apocytochrome c. They are also completely devoid of cytochrome c heme lyase activity. These defects explain the inability of the cyt-2-1 mutant to convert apocytochrome c to the holo form and to import it into mitochondria

A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation

The Neurospora crassa TOB Complex: Analysis of the Topology and Function of Tob38 and Tob37

The TOB or SAM complex is responsible for assembling several proteins into the mitochondrial outer membrane, including all β-barrel proteins. We have identified several forms of the complex in Neurospora crassa. One form contains Tob55, Tob38, and Tob37; another contains these three subunits plus the Mdm10 protein; while additional complexes contain only Tob55. As previously shown for Tob55, both Tob37 and Tob38 are essential for viability of the organism. Mitochondria deficient in Tob37 or Tob38 have reduced ability to assemble β-barrel proteins. The function of two hydrophobic domains in the C-terminal region of the Tob37 protein was investigated. Mutant Tob37 proteins lacking either or both of these regions are able to restore viability to cells lacking the protein. One of the domains was found to anchor the protein to the outer mitochondrial membrane but was not necessary for targeting or association of the protein with mitochondria. Examination of the import properties of mitochondria containing Tob37 with deletions of the hydrophobic domains reveals that the topology of Tob37 may be important for interactions between specific classes of β-barrel precursors and the TOB complex

Functions of the Small Proteins in the TOM Complex of Neurospora crasssa

The TOM (translocase of the outer mitochondrial membrane) complex of the outer mitochondrial membrane is required for the import of proteins into the organelle. The core TOM complex contains five proteins, including three small components Tom7, Tom6, and Tom5. We have created single and double mutants of all combinations of the three small Tom proteins of Neurospora crassa. Analysis of the mutants revealed that Tom6 plays a major role in TOM complex stability, whereas Tom7 has a lesser role. Mutants lacking both Tom6 and Tom7 have an extremely labile TOM complex and are the only class of mutant to exhibit an altered growth phenotype. Although single mutants lacking N. crassa Tom5 have no apparent TOM complex abnormalities, studies of double mutants lacking Tom5 suggest that it also has a minor role in maintaining TOM complex stability. Our inability to isolate triple mutants supports the idea that the three proteins have overlapping functions. Mitochondria lacking either Tom6 or Tom7 are differentially affected in their ability to import different precursor proteins into the organelle, suggesting that they may play roles in the sorting of proteins to different mitochondrial subcompartments. Newly imported Tom40 was readily assembled into the TOM complex in mitochondria lacking any of the small Tom proteins

Genetic Evidence for a Regulatory Pathway Controlling Alternative Oxidase Production in Neurospora crassa

When the cytochrome-mediated mitochondrial electron transport chain of Neurospora crassa is disrupted, an alternative oxidase encoded by the nuclear aod-1 gene is induced. The alternative oxidase donates electrons directly to oxygen from the ubiquininol pool and is insensitive to chemicals such as antimycin A and KCN that affect the standard electron transport chain. To facilitate isolation of mutants affecting regulation of aod-1, a reporter system containing the region upstream of the aod-1 coding sequence fused to the coding sequence of the N. crassa tyrosinase gene (T) was transformed into a strain carrying a null allele of the endogenous T gene. In the resulting reporter strain, growth in the presence of chloramphenicol, an inhibitor of mitochondrial translation whose action decreases the level of mitochondrial translation products resulting in impaired cytochrome-mediated respiration, caused induction of both alternative oxidase and tyrosinase. Conidia from the reporter strain were mutagenized, plated on medium containing chloramphenicol, and colonies that did not express tyrosinase were identified as potential regulatory mutants. After further characterization, 15 strains were found that were unable to induce both the reporter and the alternative oxidase. Complementation analysis revealed that four novel loci involved in aod-1 regulation had been isolated. The discovery that several genes are required for regulation of aod-1 suggests the existence of a complex pathway for signaling from the mitochondria to the nucleus and/or for expression of the gene