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.

Disruption of the gene for Hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in import of proteins into mitochondria.

The gene for Hsp30, the only known alpha-crystallin-related heat shock protein of Neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. Hsp30, which is not synthesized at 30 degrees C, associates reversibly with the mitochondria at high temperature (45 degrees C). In this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired at high temperature in a strain mutant in Hsp30. After 70 min of cell incubation at 45 degrees C, most matrix, inner membrane, and intermembrane-space proteins tested were reduced in import by about 50-70% in the mutant, as compared to wild-type cells. In contrast, assembly of selected proteins into the outer mitochondrial membrane was not reduced, except for one component of the preprotein translocase complex of the mitochondrial outer membrane. Three proteins of this complex co-immunoprecipitated with Hsp30 of wild-type cells incubated at 45 degrees C. We propose that Hsp30 interacts with the preprotein translocase of the mitochondrial outer membrane and that it chaperones the activity of one or more components of this translocase complex at high temperature.

Glucose metabolism in Neurospora is altered by heat shock and by disruption of HSP30.

We compared the metabolism of [1-13C]glucose by wild type cells of Neurospora crassa at normal growth temperature and at heat shock temperatures, using nuclear magnetic resonance analysis of cell extracts. High temperature led to increased incorporation of 13C into trehalose, relative to all other metabolites, and there was undetectable synthesis of glycerol, which was a prominent metabolite of glucose at normal temperature (30 degrees C). Heat shock strongly reduced formation of tricarboxylic acid cycle intermediates, approximately 10-fold, and mannitol synthesis was severely depressed at 46 degrees C, but only moderately reduced at 45 degrees C. A mutant strain of N. crassa that lacks the small alpha-crystallin-related heat shock protein, Hsp30, shows poor survival during heat shock on a nutrient medium with restricted glucose. An analysis of glucose metabolism of this strain showed that, unlike the wild type strain, Hsp30-deficient cells may accumulate unphosphorylated glucose at high temperature. This suggestion that glucose-phosphorylating hexokinase activity might be depressed in mutant cells led us to compare hexokinase activity in the two strains at high temperature. Hexokinase was reduced more than 35% in the mutant cell extracts, relative to wild type extracts. alpha-Crystallin and an Hsp30-enriched preparation protected purified hexokinase from thermal inactivation in vitro, supporting the proposal that Hsp30 may directly stabilize hexokinase in vivo during heat shock.

Characterization of an 88-kDa heat shock protein of Neurospora crassa that interacts with Hsp30.

The small heat shock protein of Neurospora crassa, Hsp30, when employed in affinity chromatography, bound two cellular proteins that were identified as Hsp70 and Hsp88. Both Hsp70 and Hsp88 bound to Hsp30 in preference to other proteins, but binding of Hsp88 was more selective for Hsp30, and a direct interaction was observed. Transcripts for Hsp88, a newly characterized protein, are present at normal temperature, but they are strongly induced by heat shock. Its cDNA sequence predicts a protein with homology to mammalian Hsp110 family proteins, which are distantly related to Hsp70. Hsp88 and its homologues show greater similarity to Hsp70 in its N-terminal ATPase domain than in the C-terminal peptide-binding domain, and its ATP-binding motifs are conserved. Nevertheless, the N-terminal domain of Hsp88 (and related proteins) is consistently more hydrophobic and more basic than that of Hsp70 proteins. Within the C-terminal domain, the sequence corresponding to the DnaK alpha subdomain is conserved in the Hsp88/Hsp110 family proteins, whereas the DnaK beta subdomain sequence is not conserved. The interaction between Hsp70 and Hsp30 may reflect their cooperation as cochaperones for denatured proteins, whereas Hsp88 and Hsp30 may form a complex that interacts with potential substrates.

Cytochrome c oxidase in Neurospora crassa contains myristic acid covalently linked to subunit 1.

Radiolabel from [3H]myristic acid was incorporated by Neurospora crassa into the core catalytic subunit 1 of cytochrome c oxidase (EC 1.9.3.1), as indicated by immunoprecipitation. This modification of the subunit, which was specific for myristic acid, represents an uncommon type of myristoylation through an amide linkage at an internal lysine, rather than an N-terminal glycine. The [3H]myristate, which was chemically recovered from the radiolabeled subunit peptide, modified an invariant Lys-324, based upon analyses of proteolysis products. This myristoylated lysine is found within one of the predicted transmembrane helices of subunit 1 and could contribute to the environment of the active site of the enzyme. The myristate was identified by mass spectrometry as a component of mature subunit 1 of a catalytically active, purified enzyme. To our knowledge, fatty acylation of a mitochondrially synthesized inner-membrane protein has not been reported previously.

Disruption of the gene for hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in thermotolerance.

The alpha-crystallin-related heat shock proteins are produced by all eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a nonmetabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.

Expression of mitochondrial genes in the germinating conidia of Neurospora crassa.

The germinating asexual spores (conidia) of Neurospora crassa were employed to study steps in the accumulation of transcripts of groups of mitochondrial genes, including those for peptide subunits of cytochrome c oxidase (CO), ATPase (ATP), and apocytochrome b (COB). Physically clustered groups of genes were expressed as cohorts: transcripts of the ATP8-ATP6-mtATP9-CO2 genes were almost undetectable in the dormant spores, and they accumulated rapidly as a group immediately after spore activation. Transcripts of COB and the adjacent CO1 were abundant in the dormant spores, and the dormant and germinating spores contained size forms of the COB transcripts that were not evident in vegetative cells. Polyribosomes were prepared from mitochondrial lysates, and the polyribosomal RNA was probed to identify the mRNAs of specific genes; in several instances polycistronic mRNAs were present in the polyribosomes as were the smaller end-products of the inferred transcript processing pathways. The expression of the physically dispersed genes for subunit peptides of cytochrome c oxidase appears to be regulated to the level of translation; these transcripts are accumulated in the total mitochondrial RNA with sharply different kinetics, but they appeared in the polyribosomes uniformly, their appearance correlating with the uniform synthesis of the subunit peptides. Transcripts for a previously reported non-functional mitochondrial gene, homologous to the functional nuclear gene for ATPase subunit 9, were found in the germinating spores, but were not detected in vegetative cells. These mtATP9 transcripts were also present in the polyribosomes and were apparently translated into a protein in vivo whose synthesis was insensitive to cycloheximide and detectable with an anti-ATP9 subunit antibody. Transcripts for two nuclear genes for mitochondrially localized proteins, ATP9 and CO5, were accumulated in unison and especially rapidly during spore germination.

Phylogeny of the alpha-crystallin-related heat-shock proteins.

Phylogenetic relationships were examined among 35 alpha-crystallin-related heat-shock proteins from animals, plants, and fungi. Approximately one-third of the aligned amino acids in these proteins were conserved in 74% of the proteins, and three blocks of consensus sequence were identified. Relationships were established by maximum parsimony and distance matrix analyses of the aligned amino acid sequences. The inferred phylogeny trees show the plant proteins clearly divided into three major groups that are unrelated to taxonomy: the chloroplast-localized proteins and two groups that originate from a common ancestral plant protein. The animal proteins, in contrast, branch in accordance with taxonomy, the only clear exception being the alpha-crystallin subgrouping of vertebrates. This analysis indicates that the small heat-shock proteins of animals have diverged more widely than have the plant proteins, one group of which is especially stable.

Isolation, partial amino acid sequence, and cellular distribution of heat-shock protein hsp98 from Neurospora crassa.

Hsp98 is one of the most prominent proteins synthesized during the heat-shock response of Neurospora crassa. We purified hsp98 and determined the amino acid sequence of two overlapping peptides obtained by cyanogen bromide cleavage. This 28 amino acid sequence from hsp98 has 75% homology with a region of the ClpB protein of Escherichia coli and 86% homology to a 96-kDa protein of Trypanosoma brucei. It also has 71% homology to hsp104 of Saccharomyces cerevisiae. Hsp98 was enriched in the microsomal fraction of heat-shocked cells. Sucrose gradient analysis of this cellular fraction showed that the three major high molecular weight heat-shock proteins (hsp98, 83 and 67) were more concentrated in polyribosomes than in monoribosomes. Another newly synthesized protein, p28, was strongly enriched in monoribosomes. After dissociation of the polyribosomes into ribosomal subunits, the three major heat-shock proteins were shown to be localized preferentially in the large subunit. Whereas p28 was also strongly associated with the large ribosomal subunit, a newly synthesized protein of about 22 kDa was exclusively associated with the small subunit.

The heat shock response of pollen and other tissues of maize.

While a heat shock treatment of 40 degrees C or 45 degrees C induced the vegetative tissues of maize to produce the typical heat shock proteins (HSPs), germinating maize pollen exposed to the same temperatures did not synthesize these characteristic HSPs. Comparison of RNA accumulation in shoot and tassel tissue showed that mRNAs for HSP70 and HSP18 increased several-fold, reaching high levels within 1 or 2 hours. At the higher temperature of 45 degrees C these vegetative tissues were blocked in removal of an intron from the HSP70 mRNA precursor, which accumulated to a high level in tassel tissue. In germinating pollen exposed to heat shock, mRNAs for these HSPs were induced but accumulated only to low levels. The stressed pollen maintained high levels of RNA for alpha-tubulin, a representative normal transcript. It is likely that the defective heat shock response of maize pollen is due to inefficient induction of heat shock gene transcription.

Respiration and gene expression in germinating ascospores of Neurospora tetrasperma.

Cellular proteins were not synthesized by germinating ascospores of Neurospora tetrasperma until 90 min after spore activation. Nevertheless, immediately after activation these ascospores developed a cyanide-sensitive respiration which increased throughout this 90-min period. At 90 min the respiratory rates accelerated rapidly, protein synthesis was initiated, and transcripts for a subunit of the mitochondrial ATPase, employed here as a representative mRNA, began to accumulate.

Heat shock gene expression in germinating ascospores of Neurospora tetrasperma.

The activated ascospores of Neurospora tetrasperma were inactive in protein synthesis and did not accumulate transcripts for a constitutive protein until after 90 min of incubation. These spores were blocked even longer in the expression of a gene encoding a heat shock protein, hsp30, which could not be induced until after 300 min of spore germination. Early in germination the ascospores were highly susceptible to damage from moderately high temperatures. At the same time that spores became capable of expressing the hsp30 gene, there was a loss of cytosine methylation from the gene.

The respiratory response to heat shock in Neurospora crassa.

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.

Gene sequence and analysis of hsp30, a small heat shock protein of Neurospora crassa which associates with mitochondria.

hsp30 is a small heat shock protein of Neurospora crassa which earlier studies suggested may associate with mitochondria during cellular heat shock. We show here that the association of hsp30 with mitochondria is reversible and that hsp30 dissociates after cells are returned to normal temperature. We sequenced the gene for hsp30 and defined its transcript by S1 nuclease analysis and cDNA sequencing. The gene apparently is present in the genome as a single copy, and it contains no introns. The encoded 25.3-kDa peptide is related to other small heat shock proteins, especially those from green plants. According to its deduced sequence, hsp30 can form two strongly amphiphilic alpha-helices, including one at its amino terminus. In binding assays, in vitro synthesized hsp30 bound strongly to mitochondria isolated from heat-shocked cells but not to mitochondria prepared from cells incubated at normal temperature. A mutant hsp30 peptide, deleted in the amino-terminal amphiphilic helix, bound more avidly than the full-length hsp30 to mitochondria isolated from heat-shocked cells and exhibited less stringent requirements for binding. The mutant peptide also showed strong affinity for mitochondria isolated from unstressed cells.

Respiration and mitochondrial biogenesis in germinating embryos of maize.

Function of the cyanide-sensitive mitochondrial electron transport system was required for germination of the Zea mays embryo. Respiration of the standard electron transport system (rather than the alternate oxidase) began immediately upon initiation of imbibition. This respiration depended upon cytochrome c oxidase and ATPase that were conserved in an active form in the quiescent embryo rather than upon newly synthesized or assembled enzyme complexes. Immunoprecipitation of radiolabeled subunits of these enzymes showed that the initiation of mitochondrial biogenetic activities, including de novo synthesis of nuclear- and mitochondrial-encoded enzyme subunit peptides, was strongly induced after 6 hours of embryo germination. Undetectable or very low levels of transcripts for subunits 1 and 2 of the F(1)-ATPase and subunit 2 of cytochrome c oxidase were present in the quiescent embryo; these transcripts accumulated rapidly between 6 and 12 hours of germination and their translation products were rapidly synthesized between 6 and 24 hours. An exception was the gene for subunit 9 of the ATPase; transcripts of this mitochondrial gene were abundant in the dry embryo and rapidly accumulated further upon initiation of imbibition; they were translated actively during the first 6 hours. We isolated and sequenced a near full-length cDNA for subunit 2 (beta) of the F(1)-ATPase, and we compared the deduced protein sequence with related sequences of other organisms.

Two developmental stages of Neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for recovery.

At the heat shock temperature of 45 degrees C, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of Neurospora crassa. Both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. At the RNA level, however, these two developmental stages responded with different kinetics to elevated temperature. Heat shock RNAs (for hsp30 and hsp83) accumulated and declined more rapidly in spores than in mycelia, and during recovery spores accumulated mRNA that encoded a normal protein (the proteolipid subunit of the mitochondrial ATPase), whereas mycelia showed no increase in this normal RNA (for at least 120 min). Therefore, the resumption of normal protein synthesis in spores may depend upon accumulation of new mRNAs. In contrast, mycelial cells appeared to change their translational preference during continued incubation at elevated temperature, from a discrimination against normal mRNAs to a resumption of their translation into normal cellular proteins, exemplified by the ATPase proteolipid subunit whose synthesis was measured in the heat-shocked cells.

Pontentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells : kinetin enhances mitochondrial fluorescence.

Selected fluorescent dyes were tested for uptake by mitochrondria in intact cells of barley, maize, and onion. The cationic cyanine dye 3,3'-diheptyloxacarbocyanine iodide [DiOC(7)(3)] accumulated in mitochondria within 15 to 30 minutes without appreciable staining of other protoplasmic constituents. The number, shape, and movement of the fluorescent mitochondria could be seen readily, and the fluorescence intensity of the mitochondria could be monitored with a microscope photometer. Fluorescence was eliminated in 1 to 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicating that maintenance of dye concentration was dependent on the inside-negative transmembrane potential maintained by functional mitochondria. Fluorescence of prestained mitochondria was enhanced within 5 to 10 minutes after addition of 0.1 millimolar kinetin to cells. The fluorescence in kinetintreated cells was dissipated by CCCP. These results suggest that kinetin interacted with respiratory processes resulting in higher potential across the mitochondrial membrane.

Heat shock protects germinating conidiospores of Neurospora crassa against freezing injury.

Germinating conidiospores of Neurospora crassa that were exposed to 45 degrees C, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees C. Whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. Survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies that grew on solid medium, and dry-weight accumulation during exponential growth in liquid culture. The heat shock-induced protection against freezing injury was transient. Spores transferred to normal growth temperature after exposure to heat shock and before freezing lost the heat shock-induced protection within 30 min. Spores subjected to freezing and thawing stress synthesized small amounts of the heat shock proteins that are synthesized in large quantities by cells exposed to 45 degrees C. Pulse-labeling studies demonstrated that neither chilling the spores to 10 degrees C or 0 degrees C in the absence of freezing nor warming the spores from 0 degrees C to 30 degrees C induced heat shock protein synthesis. The presence of the protein synthesis inhibitor cycloheximide during spore exposure to 45 degrees C did not abolish the protection against freezing injury induced by heat shock. Treatment of the cells with cycloheximide before freezing, without exposure to heat shock, itself increased spore survival.