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.

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.

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.

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.

Pantothenate is required in Neurospora crassa for assembly of subunit peptides of cytochrome c oxidase and ATPase/ATP synthase.

One polypeptide subunit of cytochrome c oxidase (EC 1.9.3.1) and two subunits of the ATPase/ATP synthase (EC 3.6.1.34) in mitochondria of Neurospora crassa are covalently modified with a derivative of pantothenic acid. In asexual spores of a pantothenate auxotroph of Neurospora, deprivation of pantothenic acid blocked the increase of the specific activities of cytochrome c oxidase and the ATPase above the basal activities in the dormant spores. Under cellular panthothenate deprivation, all the subunit peptides of these two enzymes apparently were synthesized and accumulated in the mitochondria, but these subunits were not assembled into normal complexes, and 55Fe-labeled heme a was incorporated into immunoprecipitable cytochrome c oxidase to a very low extent. In pantothenate-supplemented cells, the pantothenate derivative apparently is attached to the free unassembled subunits and appears not to be present in the assembled enzymes. It is likely that cellular deprivation of pantothenate, resulting in failure to modify the three subunit peptides, causes an interruption of the assembly pathway of cytochrome c oxidase and the ATPase/ATP synthase.

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative.

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.