Isolation of a gene encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe

We have isolated cDNA and genomic clones encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe. Nucleotide sequence analysis indicates that the encoded protein is homologous to the HSP70s of other organisms. The highest degree of amino acid conservation is with the proteins encoded by the Escherichia coli dnaK gene, the SSC1 gene of Saccharomyces cerevisiae and the MTP70 gene of Trypanosoma cruzi, the latter two having recently been shown to be located in the mitochondria. Western-blot analysis with immunoglobulin G raised against a peptide corresponding to the C terminus of the SSP1 protein indicates a 70-kDa protein which is associated with the mitochondria.     

Mapping of the ras1 gene of Schizosaccharomyces pombe

Summary The ras1 gene, an oncogene homologue, is known to be essential for recognition of the mating pheromone and hence for conjugation but not for vegetative growth in Schizosaccharomyces pombe. To facilitate further characterization and genetic manipulation of this gene, we have mapped it by using S. pombe strains which carry the Saccharomyces cerevisiae LEU2 gene inserted next to ras1 on the chromosome. Crosses with tester strains revealed that ras1 is tightly linked to pro2 on chromosome I. Furthermore, we have shown that ras1 is allelic with ste5, one of the sterility genes described by O. Girgsdies. The map position previously reported for ste5 eventually turned out to be false.     

The amiloride resistance gene,car1, of Schizosaccharomyces pombe

Amiloride, an inhibitor of various sodium transporters, is toxic to Schizosaccharomyces pombe at low concentration in minimal but not in rich media. Amiloride-resistant mutants were isolated and shown to represent a new locus (car1 for changed amiloride resistance) on chromosome I. The carl gene was cloned and sequenced. Sequence analysis revealed an open reading frame of 526 amino acids with a predicted molecular weight of 58 545 Da. It has 52% hydrophobic residues and belongs to the class of 12-transmembrane-domain transport proteins. Gene disruption of carl results in increased amiloride resistance. earl has sequence similarity to proteins from Candida associated with resistance to benomyl, methotrexate and cycloheximide. No single physiologically identifiable component of sodium transport appeared to be lost. We propose that earl serves an uptake function, perhaps as a symport with an unknown substrate and this carrier may transport amiloride into the cell. Further, we suggest that amiloride toxicity at low concentrations is not due to its effect on sodium transport but, rather, depends on intracellular interference with an unknown biosynthetic pathway.     

Analysis of the cps1 gene provides evidence for a septation checkpoint in Schizosaccharomyces pombe

The fission yeast gene cps1, which encodes the catalytic subunit of β-glucan synthase, was isolated in a screen for mutants that show an increase in ploidy at the restrictive temperature. cps1 mutants display defects in both polarity and septation at the permissive temperature, and become swollen and multinucleate at the restrictive temperature. Analysis of the interaction of cps1 with other mutations suggests the existence of a septation checkpoint, which requires the activity of the protein kinase wee1 for function.     

sar1, a gene from Schizosaccharomyces pombe encoding a protein that regulates ras1.

Proper ras1 function is required for normal sexual function in the yeast Schizosaccharomyces pombe. We have found a gene in S. pombe, sar1, that encodes a product capable of regulating ras1 function. sar1 is a member of an expanding family of RAS GTPase-activating proteins (GAPs) that includes mammalian GAP, the yeast Saccharomyces cerevisiae IRA proteins, and the product of the human neurofibromatosis locus, NF1 sar1, like these other proteins, can complement the loss of IRA function in S. cerevisiae. Computer analysis shows that the highest degree of sequence conservation is restricted to a very small number of diagnostic residues represented by the motif Phe-Leu-Arg-X-X-X-Pro-Ala-X-X-X-Pro. We find no evidence that sar1 is required for the effector function of ras1.     

A novel gene, msa1, inhibits sexual differentiation in Schizosaccharomyces pombe

Sexual differentiation in the fission yeast Schizosaccharomyces pombe is triggered by nutrient starvation or by the presence of mating pheromones. We identified a novel gene, msa1, which encodes a 533-aa putative RNA-binding protein that inhibits sexual differentiation. Disruption of the msa1 gene caused cells to hypersporulate. Intracellular levels of msa1 RNA and Msa1 protein diminished after several hours of nitrogen starvation. Genetic analysis suggested that the function of msa1 is independent of the cAMP pathway and stress-responsive pathway. Deletion of the ras1 gene in diploid cells inhibited sporulation and in haploid cells decreased expression of mating-pheromone-induced genes such as mei2, mam2, ste11, and rep1; simultaneous deletion of msa1 reversed both phenotypes. Overexpression of msa1 decreased activated Ras1(Val17)-induced expression of mam2. Phenotypic hypersporulation was similar between cells with deletion of only rad24 and both msa1 and rad24, but simultaneous deletion of msa1 and msa2/nrd1 additively increased hypersporulation. Therefore, we suggest that the primary function of Msa1 is to negatively regulate sexual differentiation by controlling the expression of Ste11-regulated genes, possibly through the pheromone-signaling pathway.     

Characterisation of PHSP1, a cDNA encoding a mitochondrial HSP70 fromPisum sativum

A pea cDNA clone,PHSP1, encoding a member of the HSP70 gene family has been isolated. DNA sequence analysis indicates that the protein encoded byPHSP1 is a homologue of the mitochondrial HSP70 proteins, SSP1 fromSchizosaccharomyces pombe and SSC1 fromS. cerevisiae. It contains an amino-terminal extension of 50 amino acids, rich in basic and hydroxyl amino acids, similar to other plant mitochondrial leader sequences. Western blot analysis indicates that the PHSP1 protein is associated only with mitochondria and not with any other sub-cellular organelle or cytoplasm. Further confirmation of its location within mitochondria was obtained fromin vitro protein translocation experiments into purifiedPisum sativum mitochondria. It was observed that the precursor protein was efficiently imported and that it is processed to produce a protein with anM _r of the anticipated size of the mature protein. Results are discussed with respect to the structure and function of the mitochondrial HSP70 protein.Abbreviations mtHSP70 mitochondrial HSP70 - ER endoplasmic reticulum - nt nucleotide - IgG immunoglobulin G - BiP immunoglobulin-binding protein - hsc heat shock cognate     

Polymorphism in heat-shock protein 70-1 (HSP70-1) gene promoter region and susceptibility to tuberculoid leprosy and pulmonary tuberculosis in Asian Indians

Heat shock proteins (HSP) act as immunological target structures either by themselves because of an unusual expression pattern, or they are carrier proteins for immunogenic peptides. A three-allele polymorphism of HSP70-1 promoter region was analysed in random patients with pulmonary tuberculosis (PTB), or with tuberculoid (TT) leprosy and healthy controls from North India. HSP70-1A and HSP70-1C occurred more frequently (> 60%) while HSP70-1B occurred infrequently in this population. Only HSP70-1A allele was significantly increased in TT leprosy as compared to healthy controls (91.8% Vs 71.1%, Pc < 0.03, RR = 4.58). Although a strong association of HLA-DR15 was observed with both of these patient groups in earlier studies, no correlation was found between HSP70-1 promoter alleles with any of the HLA allotypes. Amongst six possible genotype combinations of HSP70-1 promoter allele, only four (A/A, A/B, A/C, C/C) were encountered in Asian Indians. A significant increase of HSP70-1 A/C genotype was observed among DR15 negative PTB patients as compared to DR15 negative controls (87.5% Vs 35.7%, X2 = 8.6, Pc < 0.02) giving highest relative risk of 12.6. These findings suggest that HSP70-1 genes may play a secondary role to HLA-DR in governing susceptibility to mycobacterial infectious diseases.     

Mmd1p, a novel, conserved protein essential for normal mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe

The mmd1 mutation causes temperature-sensitive growth and defects in mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe. In mutant cells, mitochondria aggregate at the two cell ends, with increased aggregation at elevated temperatures. Microtubules, which mediate mitochondrial positioning in fission yeast, seem normal in mmd1 cells at permissive temperature and after several hours at the nonpermissive temperature but display aberrant organization after prolonged periods at 37 degrees C. Additionally, cells harboring both mmd1 and ban5-4, a temperature-sensitive allele of alpha2-tubulin, display synthetic defects in growth and mitochondrial distribution. The mmd1 mutation maps to an open reading frame encoding a novel 35.7-kDa protein. The Mmd1p sequence features repeating EZ-HEAT motifs and displays high conservation with uncharacterized homologues found in a variety of organisms. Saccharomyces cerevisiae cells depleted for their MMD1 homologue show increased sensitivity to the antimicrotubule drug benomyl, and the S. cerevisiae gene complemented the S. pombe mutation. Mmd1p was localized to the cytosol. Mmd1p is the first identified component required for the alignment of mitochondria along microtubules in fission yeast.