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S Subramani

Publications and source records attributed to S Subramani.

At least 55 records · Page 3Linked to original sources
PubMed1996

p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe.

All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.

CDC2 Protein Kinase↗
PubMed1996

Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris.

The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis. We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy. No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis. PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxisomal integral membrane protein. Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane. PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p. The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the peroxisomal membrane, with which it associates tightly. A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences. The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.

ATP-Binding Cassette Transporters↗
PubMed1996

Stability of invertase in reverse micelles.

The stability of invertase was studied under various conditions, including at 75 degrees C, in presence of stabilizers (sorbitol and glycerol) at 75 degrees C, and in the presence of denaturants (urea and trichloroacetic acid) at 37 degrees C in reverse micelles. Stability of the invertase in reverse micelles was found to be improved over that of the enzyme in bulk aqueous solution. Sorbitol could enhance enzyme stability as it does in the bulk aqueous system. The stabilizing effect of glycerol was reduced in reverse micelles. The denaturation pattern of urea remains unaltered. However, the denaturation effect of trichloroacetic acid has been reduced in reverse micelles.

Enzyme Stability↗
PubMed1996

Convergence of model systems for peroxisome biogenesis.

Receptors for the two peroxisomal targeting signals PTS1 and PTS2 have recently been identified in yeasts. The disparate subcellular locations of PTS receptor homologs have led to proposals of receptor shuttling between the cytosol and the peroxisomal membrane, and to the suggestion that some of these molecules may even reside normally in the peroxisomal matrix. A peroxisomal membrane protein that interacts with the PTS1 receptor in Saccharomyces cerevisiae may serve as the receptor-docking site on the organelle. The conservation of the PTS1 receptor in several yeasts has led to the cloning and characterization of the gene encoding its human homolog, PTS1R, which is mutated in a group of patients afflicted with fatal peroxisomal disorders. The identification of peroxisome assembly genes in yeasts is likely to lead to the cloning of additional human genes responsible for other generalized peroxisomal disorders. Protein unfolding is not a prerequisite for import of peroxisomal matrix proteins, suggesting novel mechanisms for the translocation of polypeptides across the peroxisomal membrane.

Biological Transport↗
PubMed1996

Double-strand break-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe.

The Saccharomyces cerevisiae HO gene and MATa cutting site were used to introduce site-specific double-strand breaks (DSBs) within intrachromosomal recombination substrates in Schizosaccharomyces pombe. The recombination substrates consisted of nontandem direct repeats of ade6 heteroalleles. DSB induction stimulated the frequency of recombinants 2000-fold. The spectrum of DSB-induced recombinants depended on whether the DSB was introduced within one of the ade6 repeats or in intervening unique DNA. When the DSB was introduced within unique DNA, over 99.8% of the recombinants lacked the intervening DNA but retained one copy of ade6 that was wild type or either one of the heteroalleles. When the DSB was located in duplicated DNA, 77% of the recombinants were similar to the deletion types described above, but the single ade6 copy was either wild type or exclusively that of the uncut repeat. The remaining 23% of the induced recombinants were gene convertants with two copies of ade6 and the intervening sequences; the ade6 heteroallele in which the DSB was induced was the recipient of genetic information. Half-sectored colonies were isolated, analyzed and interpreted as evidence of heteroduplex DNA formation. The results are discussed in terms of current models for recombination.

Chromosomes, Fungal↗
PubMed1996

Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells.

We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.

Base Sequence↗
PubMed1995

The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal.

The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.

Amino Acid Sequence↗
PubMed1995

The rad21 gene product of Schizosaccharomyces pombe is a nuclear, cell cycle-regulated phosphoprotein.

The rad21 gene of Schizosaccharomyces pombe is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth (Birkenbihl, R. P., and Subramani, S. (1992) Nucleic Acids Res. 20, 6605-6611). We show that the Rad21 protein migrates with an aberrantly slow mobility, has a thrombin cleavage site, and is multiply phosphorylated mainly at serine residues. The expression of the rad21 mRNA and the Rad21 protein is cell cycle-regulated, with the peak of mRNA and protein expression occurring near the G1 to S transition. Following translation of the protein, hypophosphorylated forms of the protein appear. However, the most phosphorylated form of Rad21 appears only later in the cell cycle (in S to G2). Analysis of the radiosensitive mutant rad21-45 revealed that the mutant protein is permanently hypophosphorylated. The Rad21 protein is nuclear during the cell cycle. The nuclear localization signal was identified in the C-terminal third of the protein. Upon repression of the Rad21 protein expressed from the repressible nmt1 promoter, the unphosphorylated and hypophosphorylated forms of Rad21 disappeared first. When the concentration of the most highly phosphorylated form of Rad21 sank under a critical level, the cells underwent aberrant mitoses. They exhibited loss of proper nuclear organization and abnormal septation.

Amino Acid Sequence↗
PubMed1995

Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders.

Two peroxisomal targeting signals, PTS1 and PTS2, are involved in the import of proteins into the peroxisome matrix. Human patients with fatal generalized peroxisomal deficiency disorders fall into at least nine genetic complementation groups. Cells from many of these patients are deficient in the import of PTS1-containing proteins, but the causes of the protein-import defect in these patients are unknown. We have cloned and sequenced the human cDNA homologue (PTS1R) of the Pichia pastoris PAS8 gene, the PTS1 receptor (McCollum, D., E. Monosov, and S. Subramani. 1993. J. Cell Biol. 121:761-774). The PTS1R mRNA is expressed in all human tissues examined. Antibodies to the human PTS1R recognize this protein in human, monkey, rat, and hamster cells. The protein is localized mainly in the cytosol but is also found to be associated with peroxisomes. Part of the peroxisomal PTS1R protein is tightly bound to the peroxisomal membrane. Antibodies to PTS1R inhibit peroxisomal protein-import of PTS1-containing proteins in a permeabilized CHO cell system. In vitro-translated PTS1R protein specifically binds a serine-lysine-leucine-peptide. A PAS8-PTS1R fusion protein complements the P. pastoris pas8 mutant. The PTS1R cDNA also complements the PTS1 protein-import defect in skin fibroblasts from patients--belonging to complementation group two--diagnosed as having neonatal adrenoleukodystrophy or Zellweger syndrome. The PTS1R gene has been localized to a chromosomal location where no other peroxisomal disorder genes are known to map. Our findings represent the only case in which the molecular basis of the protein-import deficiency in human peroxisomal disorders is understood.

Adrenoleukodystrophy↗
PubMed1995

Separation of phenotypes in mutant alleles of the Schizosaccharomyces pombe cell-cycle checkpoint gene rad1+.

The Schizosaccharomyces pombe rad1+ gene is involved in the G2 DNA damage cell-cycle checkpoint and in coupling mitosis to completed DNA replication. It is also required for viability when the cdc17 (DNA ligase) or wee1 proteins are inactivated. We have introduced mutations into the coding regions of rad1+ by site-directed mutagenesis. The effects of these mutations on the DNA damage and DNA replication checkpoints have been analyzed, as well as their associated phenotypes in a cdc17-K42 or a wee1-50 background. For all alleles, the resistance to radiation or hydroxyurea correlates well with the degree of functioning of checkpoint pathways activated by these treatments. One mutation, rad1-S3, completely abolishes the DNA replication checkpoint while partially retaining the DNA damage checkpoint. As single mutants, the rad1-S1, rad1-S2, rad1-S5, and rad1-S6 alleles have a wild-type phenotype with respect to radiation sensitivity and checkpoint functions; however, like the rad1 null allele, the rad1-S1 and rad1-S2 alleles exhibit synthetic lethality at the restrictive temperature with the cdc17-K42 or the wee1-50 mutation. The rad1-S5 and rad1-S6 alleles allow growth at higher temperatures in a cdc17-K42 or wee1-50 background than does wild-type rad1+, and thus behave like "superalleles." In most cases both chromosomal and multi-copy episomal mutant alleles have been investigated, and the agreement between these two states is very good. We provide evidence that the functions of rad1 can be dissociated into three groups by specific mutations. Models for the action of these rad1 alleles are discussed. In addition, a putative negative regulatory domain of rad1 is identified.

Alleles↗
PubMed1995

Import of stably folded proteins into peroxisomes.

By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse the single membrane of this organelle. Because the targeting signal for most peroxisomal matrix proteins is a carboxy-terminal tripeptide sequence (SKL or its variants), these proteins must remain import competent until their translation is complete. We sought to determine whether stably folded proteins were substrates for peroxisomal import. Prefolded proteins stabilized with disulfide bonds and chemical cross-linkers were shown to be substrates for peroxisomal import, as were mature folded and disulfide-bonded IgG molecules containing the peroxisomal targeting signal. In addition, colloidal gold particles conjugated to proteins bearing the peroxisomal targeting signal were translocated into the peroxisomal matrix. These results support the concept that proteins may fold in the mammalian cytosol, before their import into the peroxisome, and that protein unfolding is not a prerequisite for peroxisomal import.

Amino Acid Sequence↗
PubMed1994

The Schizosaccharomyces pombe rad1 gene consists of three exons and the cDNA sequence is partially homologous to the Ustilago maydis REC1 cDNA.

We show that the rad1 gene of Schizosaccharomyces pombe is comprised of three exons and encodes a protein of 37 kDa. A cDNA clone containing these three exons complements the sensitivity of the rad1-1 mutant to ultraviolet and gamma-radiation and to hydroxyurea. The newly identified ORF of the rad1 gene was found to exhibit partial homology to the REC1 gene of Ustilago maydis. These two genes share putative functional similarities in their respective organisms.

Amino Acid Sequence↗
PubMed1994

An upstream region of the enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase gene directs luciferase expression in liver in response to peroxisome proliferators in transgenic mice.

Peroxisome proliferators, which are structurally diverse nonmutagenic agents, induce hepatocarcinogenesis in rats and mice. Exposure to these xenobiotics leads to a rapid and coordinated transcriptional activation of the genes for the peroxisomal beta-oxidation enzyme system pathway in the liver. We have previously identified a peroxisome proliferator-responsive element in the 5'-flanking region of the rat peroxisomal hydratase/dehydrogenase (PBE) gene, the second enzyme in the beta-oxidation pathway. The peroxisome proliferator-responsive element in the PBE gene was shown to direct the induction of a luciferase reporter gene in vitro. We have now used this 3.2-kilobase 5'-flanking region of the PBE gene fused to the coding region of luciferase to generate transgenic mice. Three independent lines of transgenic mice expressed luciferase in response to ciprofibrate, a peroxisome proliferator. The induction of luciferase is specific to the liver; this agrees with the tissue-specific induction of PBE. Two other hypolipidemic drugs, nafenopin and Wy-14,643, were also capable of inducing luciferase activity in the liver. This study suggests that the PBE upstream element can be used to direct and modulate the expression of cloned genes by changing the levels of peroxisome proliferators. Also, the PBE-luciferase transgenic mouse should be an excellent model system for screening xenobiotics for potential peroxisome proliferator property.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1994

Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids required for import into peroxisomes in vivo.

Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase is a soluble matrix protein that does not end in a consensus peroxisomal targeting signal-1. The amino terminus of S. cerevisiae peroxisomal thiolase is conserved in 6 of 11 residues with the amino terminus of rat thiolase B, shown to act as a peroxisomal targeting signal-2 (Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991) EMBO J. 10, 3255-3262). Unlike mammalian peroxisomal thiolases, there is no extensive cleavage of S. cerevisiae thiolase upon import into peroxisomes. We demonstrate by in vivo expression that the amino-terminal 16 amino acids of S. cerevisiae thiolase are necessary and sufficient for targeting to peroxisomes. This result implies that yeast, like mammalian cells, can target proteins to the peroxisomal matrix by at least two different routes. We also demonstrate by targeted mutagenesis and in vivo expression of mutated thiolase genes that three amino acids conserved in the amino termini of all known thiolases are critical for efficient targeting of S. cerevisiae thiolase to peroxisomes.

Acetyl-CoA C-Acyltransferase↗