The Neurospora crassa AOD1 protein is a mitochondrial alternative oxidase that passes electrons directly from ubiquinol to oxygen. The enzyme is encoded by the nuclear aod-1 gene and is produced when the standard electron transport chain is inhibited. We previously identified eleven strains in the N. crassa single gene deletion library that were severely deficient in their ability to produce AOD1 when grown in the presence of chloramphenicol, an inhibitor of mitochondrial translation that is known to induce the enzyme. Three mutants affected previously characterized genes. In this report we examined the remaining mutants and found that the deficiency of AOD1 was due to secondary mutations in all but two of the strains. One of the authentic mutants contained a deletion of the yvh1 gene and was found to have a deficiency of aod-1 transcripts. The YVH1 protein localized to the nucleus and a post mitochondrial pellet from the cytoplasm. A zinc binding domain in the protein was required for rescue of the AOD1 deficiency. In other organisms YVH1 is required for ribosome assembly and mutants have multiple phenotypes. Lack of YVH1 in N. crassa likely also affects ribosome assembly leading to phenotypes that include altered regulation of AOD1 production.

The Neurospora crassa nuclear aod-1 gene encodes an alternative oxidase that functions in mitochondria. The enzyme provides a branch from the standard electron transport chain by transferring electrons directly from ubiquinol to oxygen. In standard laboratory strains, aod-1 is transcribed at very low levels under normal growth conditions. However, if the standard electron transport chain is disrupted, aod-1 mRNA expression is induced and the AOD1 protein is produced. We previously identified a strain of N. crassa, that produces high levels of aod-1 transcript under non-inducing conditions. Here we have crossed this strain to a standard lab strain and determined the genomic sequences of the parents and several progeny. Analysis of the sequence data and the levels of aod-1 mRNA in uninduced cultures revealed that a frameshift mutation in the flbA gene results in the high uninduced expression of aod-1 The flbA gene encodes a regulator of G protein signaling that decreases the activity of the Gα subunit of heterotrimeric G proteins. Our data suggest that strains with a functional flbA gene prevent uninduced expression of aod-1 by inactivating a G protein signaling pathway, and that this pathway is activated in cells grown under conditions that induce aod-1 Induced cells with a deletion of the gene encoding the Gα protein still have a partial increase in aod-1 mRNA levels, suggesting a second pathway for inducing transcription of the gene in N. crassa We also present evidence that a translational control mechanism prevents production of AOD1 protein in uninduced cultures.

In Neurospora crassa, blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (AOX). AOX transfers electrons directly from ubiquinol to molecular oxygen. AOX serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. The N. crassa transcription factors AOD2 and AOD5 are necessary for the expression of the AOX gene. To gain insight into the mechanism by which these factors function, and to determine if they have roles in the expression of additional genes in N. crassa, we constructed strains expressing only tagged versions of the proteins. Cell fractionation experiments showed that both proteins are localized to the nucleus under both AOX inducing and noninducing conditions. Furthermore, chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis revealed that the proteins are bound to the promoter region of the AOX gene under both conditions. ChIP-seq also showed that the transcription factors bind to the upstream regions of a number of genes that are involved in energy production and metabolism. Dependence on AOD2 and AOD5 for the expression of several of these genes was verified by quantitative PCR. The majority of ChIP-seq peaks observed were enriched for both AOD2 and AOD5. However, we also observed occasional sites where one factor appeared to bind preferentially. The most striking of these was a conserved sequence that bound large amounts of AOD2 but little AOD5. This sequence was found within a 310 bp repeat unit that occurs at several locations in the genome.

Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a beta-barrel containing 19 membrane spanning beta-trands and an N-terminal alpha-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential beta-trands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 beta-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space.

The endoplasmic reticulum mitochondria encounter structure (ERMES) tethers the er to mitochondria and contains four structural components: Mmm1, Mdm12, Mdm10, and Mmm2 (Mdm34). The Gem1 protein may play a role in regulating ERMES function. Saccharomyces cerevisiae and Neurospora crassa strains lacking any of Mmm1, Mdm12, or Mdm10 are known to show a variety of phenotypic defects including altered mitochondrial morphology and defects in the assembly of β-barrel proteins into the mitochondrial outer membrane. Here we examine ERMES complex components in N. crassa and show that Mmm1 is an ER membrane protein containing a Cys residue near its N-terminus that is conserved in the class Sordariomycetes. The residue occurs in the ER-lumen domain of the protein and is involved in the formation of disulphide bonds that give rise to Mmm1 dimers. Dimer formation is required for efficient assembly of Tom40 into the TOM complex. However, no effects are seen on porin assembly or mitochondrial morphology. This demonstrates a specificity of function and suggests a direct role for Mmm1 in Tom40 assembly. Mutation of a highly conserved region in the cytosolic domain of Mmm1 results in moderate defects in Tom40 and porin assembly, as well as a slight morphological phenotype. Previous reports have not examined the role of Mmm2 with respect to mitochondrial protein import and assembly. Here we show that absence of Mmm2 affects assembly of β-barrel proteins and that lack of any ERMES structural component results in defects in Tom22 assembly. Loss of N. crassa Gem1 has no effect on the assembly of these proteins but does affect mitochondrial morphology.

The alternative oxidase (AOX) of Neurospora crassa transfers electrons from ubiquinol to oxygen. The enzyme is not expressed under normal conditions. However, when the function of the standard electron transport chain is compromised, AOX is induced, providing cells with a means to continue respiration and growth. Induction of the enzyme represents a form of retrograde regulation because AOX is encoded by a nuclear gene that responds to signals produced from inefficiently functioning mitochondria. To identify genes required for AOX expression, we have screened the N. crassa gene knockout library for strains that are unable to grow in the presence of antimycin A, an inhibitor of complex III of the standard electron transport chain. From the 7800 strains containing knockouts of different genes, we identified 62 strains that have reduced levels of AOX when grown under conditions known to induce the enzyme. Some strains have virtually no AOX, whereas others have only a slight reduction of the protein. A broad range of seemingly unrelated functions are represented in the knockouts. For example, we identified transcription factors, kinases, the mitochondrial import receptor Tom70, three subunits of the COP9 signalosome, a monothiol glutaredoxin, and several hypothetical proteins as being required for wild-type levels of AOX production. Our results suggest that defects in many signaling or metabolic pathways have a negative effect on AOX expression and imply that complex systems control production of the enzyme.

Early in their formation, memories are thought to be labile, requiring a process called consolidation to give them near-permanent stability. Evidence for consolidation as an active and biologically separate mnemonic process has been established through posttraining manipulations of the brain that promote or disrupt subsequent retrieval. Consolidation is thought to be ultimately mediated via protein synthesis since translational inhibitors such as anisomycin disrupt subsequent memory when administered in a critical time window just following initial learning. However, when applied intracerebrally, they may induce additional neural disturbances. Here, we report that intrahippocampal microinfusions of anisomycin in urethane-anesthetized rats at dosages previously used in memory consolidation studies strongly suppressed (and in some cases abolished) spontaneous and evoked local field potentials (and associated extracellular current flow) as well as multiunit activity. These effects were not coupled to the production of pathological electrographic activity nor were they due to cell death. However, the amount of suppression was correlated with the degree of protein synthesis inhibition as measured by autoradiography and was also observed with cycloheximide, another translational inhibitor. Our results suggest that (1) the amnestic effects of protein synthesis inhibitors are confounded by neural silencing and that (2) intact protein synthesis is crucial for neural signaling itself.

The TOB-SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55-Sam50, Tob38-Sam35, and Tob37-Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB-SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.

We present clinical, neuroimaging, and molecular data on the identification of a new homozygous c.1783A>G (p.Thr595Ala) mutation in NDUFS1 in two inbred siblings with isolated complex I deficiency associated to a progressive cavitating leukoencephalopathy, a clinical and neuroradiological entity originally related to unknown defects of the mitochondrial energy metabolism. In both sibs, the muscle biopsy showed severe reduction of complex I enzyme activity, which was not obvious in fibroblasts. We also observed complex I dysfunction in a Neurospora crassa model of the disease, obtained by insertional mutagenesis, and in patient fibroblasts grown in galactose. Altogether, these results indicate that the NDUFS1 mutation is responsible for the disease and complex I deficiency. Clinical presentation of complex I defect is heterogeneous and includes an ample array of clinical phenotypes. Expanding the number of allelic variants in NDUFS1 , our findings also contribute to a better understanding on the function of complex I.

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.

This chapter covers the functions of mitochondria in filamentous fungi. Mitochondrial respiration occurs via transfer of electrons from reduced electron carriers to molecular oxygen. This is accomplished using an electron transport chain housed in the mitochondrial inner membrane (MIM). Fungal mitochondrial genomes are AT-rich DNAs that map as circular molecules. However, several studies suggest that fungal mtDNAs are actually long, concatenated linear molecules. The study of fungal mitochondrial introns has been an active area of research that has yielded a number of significant findings. There are numerous reports of RNA elements being associated with mitochondria. Though they are formally classified as mitoviruses, they can also be considered plasmids based on their structural similarities to linear mt plasmids and lack of an extracellular form. The large number of different proteins that are found in mitochondria (probably about 1,000 for fungi) and the limited coding capacity of mtDNA emphasize the fact that most mitochondrial proteins are encoded by nuclear genes, translated in the cytosol, and imported into the organelle. The chapter attempts to define the basic import process and summarizes the literature while pointing out contributions that have involved filamentous fungi-almost entirely N. crassa. Filamentous fungi are generally considered immortal. Podospora anserina is an exception to this rule, as it has a defined vegetative- growth life span.

The Mdm10, Mdm12, and Mmm1 proteins have been implicated in several mitochondrial functions including mitochondrial distribution and morphology, assembly of beta-barrel proteins such as Tom40 and porin, association of mitochondria and endoplasmic reticulum, and maintaining lipid composition of mitochondrial membranes. Here we show that loss of any of these three proteins in Neurospora crassa results in the formation of large mitochondrial tubules and reduces the assembly of porin and Tom40 into the outer membrane. We have also investigated the relationship of Mdm10 and Tom7 in the biogenesis of beta-barrel proteins. Previous work showed that mitochondria lacking Tom7 assemble Tom40 more efficiently, and porin less efficiently, than wild-type mitochondria. Analysis of mdm10 and tom7 single and double mutants, has demonstrated that the effects of the two mutations are additive. Loss of Tom7 partially compensates for the decrease in Tom40 assembly resulting from loss of Mdm10, whereas porin assembly is more severely reduced in the double mutant than in either single mutant. The additive effects observed in the double mutant suggest that different steps in beta-barrel assembly are affected in the individual mutants. Many aspects of Tom7 and Mdm10 function in N. crassa are different from those of their homologues in Saccharomyces cerevisiae.

Alternative oxidase (AOX) has been found in a large number of filamentous fungi and yeasts with the notable exceptions of Saccharomyces cerevisiae and Schizosaccharomyces pombe. In virtually all of these fungi, AOX is induced by stresses on the cell that compromise the efficiency of the standard mitochondrial electron transport chain. As AOX is encoded in the nucleus and the signals that induce its expression originate in mitochondria, induction of the enzyme provides a classic example of retrograde regulation where signals from mitochondria influence the expression of nuclear genes. We have previously isolated mutants in Neurospora crassa that are incapable of inducing AOX. The genes affected in two of these mutants, aod-2 and aod-5, encode zinc cluster transcription factors that act to control expression of the AOX by binding to an alternative oxidase induction motif (AIM) found in the promoter of the AOX structural gene. We have now used pull-down assays and size-exclusion chromatography to demonstrate that the AOD2 and AOD5 proteins physically interact in vitro. In addition, we have shown that a homolog of the RTG2 protein, which is part of a classic retrograde signaling pathway in S. cerevisiae, is not required for AOX regulation in N. crassa.

Tob55 is the major component of the TOB complex, which is found in the outer membrane of mitochondria. A sheltered knockout of the tob55 gene was developed in Neurospora crassa. When grown under conditions that reduce the levels of the Tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import β-barrel proteins. Surprisingly, Western blots of wild-type mitochondrial proteins revealed two bands for Tob55 that differed by ∼4 kDa in their apparent molecular masses. Sequence analysis of cDNAs revealed that the tob55 mRNA is alternatively spliced and encodes three isoforms of the protein, which are predicted to contain 521, 516, or 483 amino acid residues. Mass spectrometry of proteins isolated from purified outer membrane vesicles confirmed the existence of each isoform in mitochondria. Strains that expressed each isoform of the protein individually were constructed. When cells expressing only the longest form of the protein were grown at elevated temperature, their growth rate was reduced and mitochondria isolated from these cells were deficient in their ability to assembly β-barrel proteins.

The alternative oxidase transfers electrons from ubiquinol to molecular oxygen, providing a mechanism for bypassing the later steps of the standard cytochrome-mediated electron transport chain. The enzyme is found in an array of organisms and in many cases is known to be produced in response to perturbations of the standard chain. Alternative oxidase is encoded in the nucleus but functions in the inner mitochondrial membrane. This implies the existence of a retrograde regulation pathway for communicating from the mitochondrion to the nucleus to induce alternative oxidase expression. Previous studies on alternative oxidase in fungi and plants have shown that a number of genes are required for expression of the enzyme, but the identity of these genes has remained elusive. By gene rescue we have now shown that the aod-2 and aod-5 genes of Neurospora crassa encode transcription factors of the zinc-cluster family. Electrophoretic mobility shift assays show that the DNA-binding domains of the AOD2 and AOD5 proteins act in tandem to bind a sequence element in the alternative oxidase gene promoter that is required for expression. Both proteins contain potential PAS domains near their C terminus, which are found primarily in proteins involved in signal transduction.