The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA

SSH1, a newly identified member of the heat shock protein (hsp70) multigene family of the budding yeast Saccharomyces cerevisiae, encodes a protein localized to the mitochondrial matrix. Deletion of the SSH1 gene results in extremely slow growth at 23 degrees C or 30 degrees C, but nearly wild-type growth at 37 degrees C. The matrix of the mitochondria contains another hsp70, Ssc1, which is essential for growth and required for translocation of proteins into mitochondria. Unlike SSC1 mutants, an SSH1 mutant showed no detectable defects in import of several proteins from the cytosol to the matrix compared to wild type. Increased expression of Ssc1 partially suppressed the cold- sensitive growth defect of the SSH1 mutant, suggesting that when present in increased amounts, Ssc1 can at least partially carry out the normal functions of Ssh1. Spontaneous suppressors of the cold-sensitive phenotype of an SSH1 null mutant were obtained at a high frequency at 23 degrees C, and were all found to be respiration deficient. 15 of 16 suppressors that were analyzed lacked mitochondrial DNA, while the 16th had reduced amounts. We suggest that Ssh1 is required for normal mitochondrial DNA replication, and that disruption of this process in ssh1 cells results in a defect in mitochondrial function at low temperatures.     

Ty Element-Induced Temperature-Sensitive Mutations of Saccharomyces Cerevisiae

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.     

Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.     

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.     

Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.

Inactivation of the yeast nuclear gene YMe2 causes an increased rate of DNA escape from mitochondria to the nucleus. Mutations in yme2 also show genetic interactions with yme1, a second gene that affects DNA escape from mitochondria to the nucleus. The yme1 cold-sensitive growth phenotype is suppressed by yme2 mutations. In addition, yme1 yme2 double mutants exhibit a synthetic growth defect on ethanol-glycerol medium at 30 degrees C. YME2 was isolated by complementation of the synthetic growth defect of yme1 yme2 strains and was found to be identical with the previously cloned RNA12 gene. The dominant temperature-sensitive mutation RNA12-1 prevents growth of yeast cells at 37 degrees C. YME2 encodes a protein with a predicted molecular weight of 96,681 and is an integral inner mitochondrial membrane protein. The larger carboxyl-terminal domain of the YME2 gene product faces the intermembrane space. Null alleles of yme2 display the same genetic interactions with yme1 and high rate of DNA escape from mitochondria as do the originally isolated yme2 mutant strains. Disruption of yme2 causes a strain-dependent growth defect on nonfermentable carbon sources.     

Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene

To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C. We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7- 1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p.     

Mas37p, a novel receptor subunit for protein import into mitochondria

By screening a collection of Saccharomyces cerevisiae mutants temperature sensitive for growth on a nonfermentable carbon source, we have isolated a gene (termed MAS37) which encodes a novel receptor for protein import into mitochondria. Mas37p is a 37-kD outer membrane protein with two putative membrane-spanning regions. Inactivation of the MAS37 gene renders cells temperature-sensitive for respiration- driven growth, inhibits import of precursors into isolated mitochondria, and is synthetically lethal with a deletion of one of the genes encoding the import receptors Mas70p or Mas20p. Inactivation of Mas37p with specific antibodies inhibits import of different precursors to different extents; the precursor specificity of Mas37p resembles that of the previously described import receptor Mas70p. Mas70p and Mas37p form a 1:1 complex in detergent extracts of mitochondria and overexpression of one protein enhances that of the other. We suggest that the Mas37p/Mas70p heterodimer functions as a receptor for protein import into yeast mitochondria and that the mitochondrial receptor system consists of hetero-oligomeric subcomplexes with distinct binding activities, but overlapping precursor specificities.     

Biosynthesis and secretion of several enzymes in Escherichia coli dnaK and dnaJ mutants

Escherichia coli null dnaJ and dnaKdnaJ mutants were defective in the biosynthesis and secretion of several enzymes. The synthesis of beta-galactosidase induced in delta dnaJ and delta dnaKdnaJ mutants was abolished at 42 degrees C and significantly decreased at 30 and 37 degrees C. The activity of alkaline phosphatase in the periplasm in both mutant strains at high temperature was lower than in the wild-type strain. The synthesis of b-type cytochromes was defective in two deletion mutants while the synthesis of nitrate reductase-A at 42 degrees C was influenced by dnaK mutation only. The lack of DnaK and DnaJ does not impair the activity of catechol 2,3-dioxygenase irrespective of growth temperature.     

The Escherichia coli DjlA and CbpA proteins can substitute for DnaJ in DnaK-mediated protein disaggregation

The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37 degrees C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42 degrees C.     

Yeast cell death caused by mutation of the OST2 gene encoding the epsilon-subunit of Saccharomyces cerevisiae oligosaccharyltransferase

An essential epsilon-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 degrees C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

Further characterization of recessive suppression in yeast. Isolation of the low-temperature sensitive mutant of Saccharomyces cerevisiae defective in the assembly of 60 S ribosomal subunit

It has been shown that recessive suppressor mutations in the yeast Saccharomyces cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10 degrees C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P-2156, was studied in detail. After a prolonged period of incubation (70 h) under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.     

Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.     

Role of trehalose in growth at high temperature of Salmonella enterica serovar Typhimurium

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of Salmonella enterica serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45 degrees C. One of these mutations proved to be in the seqA gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with pgm (which encodes phosphoglucomutase), it is likely to be polar on the expression of the pgm gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the seqA::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an otsA mutation, which inactivates the first step of the trehalose biosynthetic pathway. The otsA mutation caused a growth defect at 45 degrees C in minimal medium containing 0.2 M NaCl that was similar to that caused by the pgm mutation, but otsA did not affect growth rate in this medium at 37 degrees C. These results suggest that the growth defect of the seqA-pgm mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45 degrees C had a fivefold higher trehalose pool size than cells grown at 30 degrees C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.     

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.