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.     

Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.     

Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA.     

The Oxa2 protein of Neurospora crassa plays a critical role in the biogenesis of cytochrome oxidase and defines a ubiquitous subbranch of the Oxa1/YidC/Alb3 protein family

Proteins of the Oxa1/YidC/Alb3 family mediate the insertion of proteins into membranes of mitochondria, bacteria, and chloroplasts. Here we report the identification of a second gene of the Oxa1/YidC/Alb3 family in the genome of Neurospora crassa, which we have named oxa2. Its gene product, Oxa2, is located in the inner membrane of mitochondria. Deletion of the oxa2 gene caused a specific defect in the biogenesis of cytochrome oxidase and resulted in induction of the alternative oxidase (AOD), which bypasses the need for complex IV of the respiratory chain. The Oxa2 protein of N. crassa complements Cox18-deficient yeast mutants suggesting a common function for both proteins. The oxa2 sequence allowed the identification of a new subfamily of Oxa1/YidC/Alb3 proteins whose members appear to be ubiquitously present in mitochondria of fungi, plants, and animals including humans.     

Cytochrome oxidase subunit III gene in Neurospora crassa mitochondria. Location and sequence

We have located and sequenced the gene for cytochrome oxidase subunit III (CoIII) in Neurospora crassa mitochondria. The CoIII gene is located downstream from the small rRNA gene within a cluster of tRNA genes and is coded by the same strand as the tRNA and the rRNA genes. Like the tRNA and the rRNA genes, the CoIII gene is also flanked by the GC-rich palindromic DNA sequences which are highly conserved in N. crassa mitochondria. The CoIII coding sequence predicts a protein 269 amino acids long including 8 tryptophan residues. All 8 tryptophan residues are coded for by UGA. This supports our previous conclusion based on the anticodon sequence of N. crassa mitochondrial tryptophan tRNA and provides evidence for the notion that use of UGA as a codon for tryptophan rather than chain termination may be a feature common to most mitochondrial protein synthesis systems. The close correspondence between the amino acid composition of N. crassa CoIII and that of the protein predicted by the CoIII gene sequence suggests that unlike in mammalian mitochondria, AUA is a codon for isoleucine and not for methionine in N. crassa mitochondria. The N. crassa CoIII sequence shows strong homologies to the corresponding yeast and human proteins (53 and 47%, respectively). The overall hydrophobic character of the protein is consistent with suggestions that most of CoIII is embedded in the mitochondrial inner membrane.     

Minimal promoter for the NAD+-specific glutamate dehydrogenase gene of Neurospora crassa.

The expression of the NAD+-specific glutamate dehydrogenase (NAD-GDH) gene of Neurospora crassa is subject to catabolite repression. To identify the minimal sequence necessary for promoter function, the 5'-flanking region of the NAD-GDH gene was screened for potential protein-binding sites. Fragments of DNA, containing sequences upstream from the ATG initiation codon, were employed as probes of Southwestern blots of total cellular protein from cells grown in media promoting repression and induction of NAD-GDH. Two polypeptides interacted differentially with a promoter probe; one was present in greater abundance in repressed cells and a higher relative level of the second was witnessed in induced cells. Electrophoretic mobility shift assays with labeled promoter fragments exhibited preferential interaction with proteins in the induced cultures. The upstream sequence containing the putative protein-binding sites was fused with the coding sequence of the green fluorescent protein (GFP). The resulting plasmid was introduced into the microconidia of an albino mutant of N. crassa by electroporation. Stable integration of the plasmid and_expression of GFP in the hyphae and conidia of the transformants were demonstrated by Southern and Western blot analysis and fluorescence microscopy.     

Molecular characterization of mutations of nlt-4, the pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa

The nit-4 genes of three conventional Neurospora crassa mutations and of the closely related species, Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the three nit-4 mutants, alleles 15, 1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of a nit-4 mutant (allele 2994) and of N. intermedia have been determined and compared with that of wild-type N. crassa. The molecular characteristics confirm that the mutated gene of 2994 originated from N. intermedia and was introgressed into N. crassa. The polyglutamine domains of the N. crassa wild type, the 2994 mutant, or N. intermedia cannot replace an upstream glutamine-rich domain which is essential for nit-4 function.     

The Oxa1 protein forms a homooligomeric complex and is an essential part of the mitochondrial export translocase in Neurospora crassa

The Oxa1 protein is a ubiquitous constituent of the inner membrane of mitochondria. Oxa1 was identified in yeast as a crucial component of the protein export machinery known as the OXA translocase, which facilitates the integration of proteins from the mitochondrial matrix into the inner membrane. We have identified the Neurospora crassa Oxa1 protein which shows a sequence identity of 22% to the yeast homologue. Despite the low level of identity, the function of the homologues is conserved as the N. crassa gene fully complemented a yeast null mutant. Genetic analysis revealed that Oxa1 is essential for viability in N. crassa. Cells propagated under conditions that severely reduce Oxa1 levels grew extremely slowly and were deficient in subunits of complex I and complex IV. Isolation of the Oxa1 complex from N. crassa mitochondria revealed a 170-180-kDa complex that contained exclusively Oxa1. Since the Oxa1 monomer has a molecular weight of 43,000, our data suggest that the OXA translocase consists of a homooligomer most likely containing four Oxa1 subunits.     

NADH dehydrogenase in Neurospora crassa contains myristic acid covalently linked to the ND5 subunit peptide

The mitochondrial, proton-pumping NADH:ubiquinone oxidoreductase consists of at least 35 subunits whose synthesis is divided between the cytosol and mitochondria; this complex I catalyzes the first steps of mitochondrial electron transfer and proton translocation. Radiolabel from [(3)H]myristic acid was incorporated by Neurospora crassa into the mitochondrial-encoded, approximately 70 kDa ND5 subunit of NADH dehydrogenase, as shown by immunoprecipitation. This myristate apparently was linked to the peptide through an amide linkage at an invariant lysine residue (Lys546), based upon analyses of proteolysis products. The myristoylated lysine residue occurs in the predicted transmembrane helix 17 (residues 539-563) of ND5. A consensus amino acid sequence around this conserved residue exists in homologous subunits of NADH dehydrogenase. Cytochrome c oxidase subunit 1, in all prokaryotes and eukaryotes, contains this same consensus sequence surrounding the lysine which is myristoylated in N. crassa.     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Molecular cloning of the L-amino-acid oxidase gene from Neurospora crassa

The addition of D-phenylalanine to starved cultures of Neurospora crassa leads to de novo synthesis of L-amino-acid oxidase. Poly(A) RNA from D-phenylalanine-treated mycelium was therefore used to generate a cDNA library which was subsequently screened by hybrid-selected translation. A positive L-amino-acid oxidase clone served as a probe to isolate the complete gene from a genomic library of N. crassa. The nucleotide sequence obtained revealed an open reading frame coding for a protein of 695 amino acids. A comparison of the deduced primary structure with the partial amino-terminal sequence of the isolated enzyme showed that the protein is synthesized as a precursor. The proform exceeds the mature enzyme by 129 amino acids. The presence of a cluster of basic amino acid residues preceding Ala129 in the precursor suggests a post-translational modification brought about by limited proteolysis. N. crassa L-amino-acid oxidase shares a highly conserved region with many well-characterized flavoproteins that is known to constitute part of the flavin-adenine dinucleotide-binding site.     

Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa

Two metal response elements, flanking an antioxidant response element, were identified in regions upstream (-3730 bp) to copper metallothionein (CuMT) gene of Neurospora crassa. Presence of copper in culture media, but not of pro-oxidants like H2O2 or menadione, induced CuMT gene expression that could not be completely abolished by antioxidants such as N-acetyl cysteine and ascorbic acid. Gel shift assays revealed the ability of nuclear extracts from copper induced cultures to bind PCR-amplified metal response or antioxidant response elements. Similar observations could not be made with cultures exposed either to pro-oxidants or antioxidants. These results differentiate between CuMT gene induction by copper from antioxidant functions associated with the identified upstream elements.