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S Subramani

Publications and source records attributed to S Subramani.

At least 19 recordsLinked to original sources
PubMed2005

Force-frequency relation in frog-ventricle is dependent on the direction of sodium/calcium exchange in diastole.

AIM: Force of contraction increases with stimulus-frequency in mammalian and amphibian hearts under control conditions. Here, we have analysed the mechanism of the force-frequency relation (FFR) in frog-ventricle. METHODS: Circular strips of frog-ventricle were subjected to field-stimulation with frequencies in the range 0.03-0.2 Hz and force recorded on a chart-recorder. In another protocol, varying rest-periods were imposed while the preparation beat steadily at 0.2 Hz and the effect of rest on post-rest beat amplitude was noted. RESULTS: Under control conditions, a positive FFR and a rest-induced decay of contraction amplitude (RID) were seen in the frequency range 0.03-0.2 Hz. With cadmium, nifedipine, nickel (40 micromol L(-1)), ryanodine and adrenaline (all drugs at 10 micromol L(-1) concentration, except nickel), the positive FFR and RID seen under control conditions persisted. When the bathing solution contained ouabain (10 micromol L(-1)) or low external sodium (40 mmol L(-1)), or high external calcium (5 mmol L(-1)), the FFR turned negative in the frequency range stated above and there were rest-induced potentiations (RIP). CONCLUSION: When the conditions favour a net leak of calcium in diastole from intracellular stores via the calcium-extrusive mode of sodium-calcium exchanger (NCX), FFR is positive. An increase in frequency lessens the diastolic interval and therefore the diastolic calcium leak, thereby augmenting force. On the other hand, interventions which favour the calcium-acquisitive mode of NCX during diastole, changed the pattern of RID to RIP and converted FFR from positive to negative. With net diastolic calcium uptake, there is better store-filling and therefore higher force at lower frequencies.

Animals↗
PubMed2001

Genomic organization, chromosomal localization and tissue specific expression of the murine Pxmp2 gene encoding the 22 kDa peroxisomal membrane protein (Pmp22).

Peroxisomes are subcellular organelles with important functions in lipid metabolism that are found in virtually all eucaryotic cells. The peroxisomal membrane contains a number of integral and peripheral membrane proteins involved in the import of peroxisomal matrix proteins and the transport of metabolites across the membrane. The most abundant peroxisomal membrane protein (Pmp) in rat peroxisomes is Pmp22, a 22 kDa protein of unknown function that is encoded by the Pxmp2 gene. To investigate the function of the Pxmp2 gene, we have initiated mouse knockout studies. The expression level of the Pxmp2 mRNA in mice was investigated by Northern blot analysis. Pxmp2 RNA was shown to be differentially expressed with highest expression levels in liver, kidney and in heart tissue. Comparison with other peroxisomal marker genes revealed that the expression of Pxmp2, Pmp70 (Pxmp1) and catalase was regulated independently. Using 5' and 3' RACE we have cloned the full-length cDNA of murine Pxmp2 which comprises 863 nucleotides and have isolated a genomic clone containing the entire murine Pxmp2. We have analyzed the complete intron/exon structure of the Pxmp2 gene which contains five exons spanning about 11 kb on the genomic clone. All intron/exon splice junctions conform to the GT/AG rule. Sequence analysis of the Pxmp2 5' flanking region revealed that it was devoid of a TATA box, but characteristic promoter elements were identified within 250 base pairs upstream of the transcriptional start site. Using a mouse/hamster radiation hybrid panel, Pxmp2 was localized on mouse chromosome 5 at 59 cM.

Animals↗
PubMed2001

The human peroxisomal targeting signal receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the cytosol.

Peroxisomal targeting signals (PTSs) are recognized by predominantly cytosolic receptors, Pex5p and Pex7p. The fate of these PTS receptors following their interactions on the peroxisomal membrane with components of docking and putative translocation complexes is unknown. Using both novel and multiple experimental approaches, we show that human Pex5p does not just bind cargo and deliver it to the peroxisome membrane, but participates in multiple rounds of entry into the peroxisome matrix and export to the cytosol independent of the PTS2 import pathway. This unusual shuttling mechanism for the PTS1 receptor distinguishes protein import into peroxisomes from that into most other organelles, with the exception of the nucleus.

Cell Fractionation↗
PubMed2001

Quantitative analysis of peroxisomal protein import in vitro.

Protein import into the peroxisome matrix is mediated by peroxisome-targeting signals (PTSs). We have developed a novel, quantitative, in vitro assay for measuring peroxisomal import of PTS1-containing proteins. This enzyme-linked immunosorbent assay-based system utilizes semi-intact human A431 cells or fibroblasts and a biotinylated version of the PTS1-containing import substrate, luciferase. We show that biotinylated luciferase accumulated in peroxisomes in a time- and temperature-dependent fashion, in a reaction stimulated by exogenously added ATP, cytosol, and zinc. No import was detected in fibroblasts from a human patient belonging to complementation group 2, who suffered from the fatal peroxisomal disorder Zellweger syndrome and lacked a functional PTS1 receptor, Pex5p. Also, the reaction was significantly inhibited by antibodies to the zinc-finger protein, Pex2p. Several lines of evidence demonstrate that biotinylated luciferase was imported into the lumen of bona fide peroxisomes. (a) Biochemical fractionation of cells after the import reaction showed a time-dependent accumulation of the import substrate within intracellular organelles. (b) Confocal fluorescence microscopy indicated that imported biotinylated luciferase colocalized with the peroxisomal protein PMP70. (c) Visualization of the imported biotinylated luciferase by indirect fluorescence or indirect immunofluorescence required disruption of the peroxisomal membrane, indicating true import rather than binding to the outside of the organelle.

Amino Acid Motifs↗
PubMed2001

Pichia pastoris Pex14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins.

The peroxisomal protein import machinery plays a central role in the assembly of this organelle in all eukaryotes. Genes encoding components of this machinery, termed peroxins or Pex proteins, have been isolated and characterized in several yeast species and in mammals, including humans. Here we report on one of these components, Pex14p, from the methylotrophic yeast Pichia pastoris. Work in other organisms has shown that Pex14p is located on the cytoplasmic surface of the peroxisomal membrane and binds peroxisomal targeting signal (PTS) receptors carrying proteins bound for the peroxisomal matrix, results that have led to the hypothesis that Pex14p is a receptor-docking protein. P. pastoris Pex14p (PpPex14p) behaves like an integral membrane protein, with its C-terminus exposed on the cytosolic side of the peroxisomal membrane. PpPex14p complexes with many peroxins, including Pex3p (Snyder et al., 1999b), Pex5p, Pex7p, Pex13p, Pex17p, itself, and a previously unreported peroxin, Pex8p. A portion of Pex14p is phosphorylated, but both phosphorylated and unphosphorylated forms of Pex14p interact with several peroxins. The interactions between Pex14p and other peroxins provide clues regarding the function of Pex14p in peroxisomal protein import.

Amino Acid Sequence↗
PubMed2001

Self-destruction in the line of duty.

Three cellular processes, microautophagy, macroautophagy, and the cytoplasm-to-vacuole (Cvt) pathway, are involved in the cargo delivery from the cytosol to the vacuole or lysosome. Recent findings have identified Cvt19 at the receptor for specific cargo binding in the Cvt pathway.

Autophagy↗
PubMed2001

Identification of a novel human peroxisomal 2,4-dienoyl-CoA reductase related protein using the M13 phage protein VI phage display technology.

Recently, we reported the successful use of the gVI-cDNA phage display technology to clone cDNAs coding for novel peroxisomal enzymes by affinity selection using immobilized antisera directed against peroxisomal subfractions (Fransen, M.; Van Veldhoven, P.P.; Subramani, S. Biochem. J., 1999, 340, 561-568). To identify other unknown peroxisomal enzymes, we further exploited this promising approach. Here we report the isolation and cloning of another novel human cDNA encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal targeting signal (PTS1). Primary structure analysis revealed that this molecule shared the highest sequence similarity to members of the 2,4-dienoyl-CoA reductase (DCR) family. However, functional analysis indicated that a recombinantly expressed version of the novel protein did not possess DCR activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA as a substrate. The recombinant protein interacted with HsPex5p, the human PTS1-binding protein. Binding was competitively inhibited by a PTS1-containing peptide and was abolished when the last amino acid of the PTS1 signal was deleted. Transfection of mammalian cells with gene fusions between green fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal localization and, therefore, the functionality of the PTS1. These results further demonstrate the suitability of the gVI-cDNA phage display technology for cDNA expression cloning using an antibody as a probe.

Amino Acid Sequence↗
PubMed2000

The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane.

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.

Binding Sites↗
PubMed2000

Import of peroxisomal matrix and membrane proteins.

This review summarizes the progress made in our understanding of peroxisome biogenesis in the last few years, during which the functional roles of many of the 23 peroxins (proteins involved in peroxisomal protein import and peroxisome biogenesis) have become clearer. Previous reviews in the field have focussed on the metabolic functions of peroxisomes, aspects of import/biogenesis the role of peroxins in human disease, and involvement of the endoplasmic reticulum in peroxisome membrane biogenesis as well as the degradation of this organelle. This review refers to some of the earlier work for the sake of introduction and continuity but deals primarily with the more recent progress. The principal areas of progress are the identification of new peroxins, definition of protein-protein interactions among peroxins leading to the recognition of complexes involved in peroxisomal protein import, insight into the biogenesis of peroxisomal membrane proteins, and, of most importance, the elucidation of the role of many conserved peroxins in human disease. Given the rapid progress in the field, this review also highlights some of the unanswered questions that remain to be tackled.

Biological Transport, Active↗
PubMed2000

Environmental response of yeast peroxisomes. Aspects of organelle assembly and degradation.

Nutritional changes can affect either the assembly or disassembly of yeast peroxisomes. In the past decade, insights regarding the molecular mechanisms of peroxisome assembly have been gained chiefly through the cloning of the PEX genes obtained by complementation of corresponding pex mutants in several yeast strains and Chinese hamster ovary cell lines. Depletion of these peroxins (proteins encoded by PEX genes) by deletion of the corresponding genes affects peroxisomal protein import, biogenesis, or proliferation. To complement these studies in the field, the authors undertook an investigation of the functions of a subset of Candida boidinii peroxisomal membrane proteins (PMPs), Pex11, Pmp47, and Pmp20, by analyzing strains of C. boidinii in which the genes encoding these proteins were deleted. The authors' studies show that Pex11p is involved in peroxisome proliferation; Pmp47 plays a role in the translocation, folding, or assembly of dihydroxyacetone synthase; and Pmp20 is probably involved in methanol metabolism. In contrast to the studies on peroxisome assembly, the molecular mechanisms of peroxisome degradation remain poorly understood. To shed light on this problem, the authors isolated Pichia pastoris mutants defective in peroxisome autophagy (pag mutants). A novel, double-fluorescence method used for the characterization of wild-type cells and of pag mutants enabled us to dissect the microautophagic degradation of peroxisomes into several distinct stages. These studies show that specific PAG gene products are involved in multiple steps of the process. Future cloning and characterization of the functions of PAG genes will reveal the molecular basis of peroxisome degradation.

Animals↗
PubMed1999

Characterization of human and murine PMP20 peroxisomal proteins that exhibit antioxidant activity in vitro.

We have isolated the cDNAs encoding human and mouse homologues of a yeast protein, termed peroxisomal membrane protein 20 (PMP20). Comparison of the amino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins revealed a high degree of identity (93%), whereas resemblance to the yeast Candida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% identity). Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B. HsPMP20 mRNA expression was low in human fetal tissues, especially in the brain. In adult tissues, HsPMP20 mRNA was expressed in the majority of tissues tested. HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes. HsPMP20 bound directly to the human peroxisomal targeting signal 1 receptor, HsPEX5. Mutagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5. Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes. Double-staining immunofluorescence studies showed colocalization of HsPMP20 and thiolase, a bona fide peroxisomal protein. The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high similarity to thiol-specific antioxidant proteins. HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catalyzed oxidation system but not in the nonthiol metal-catalyzed oxidation system, suggesting that HsPMP20 possesses thiol-specific antioxidant activity. In addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase activity. These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxidative stress. Because some portion of PMP20 might also be present in the cytosol, HsPMP20 may also have a protective effect in the cytoplasm.

Amino Acid Sequence↗
PubMed1999

Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane.

We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

Amino Acid Sequence↗
PubMed1999

Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase.

To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.

Alcohol Dehydrogenase↗
PubMed1999

Speedy: a novel cell cycle regulator of the G2/M transition.

Stage VI Xenopus oocytes are suspended at the G2/M transition of meiosis I, and represent an excellent system for the identification and examination of cell cycle regulatory proteins. Essential cell cycle regulators such as MAPK, cyclins and mos have the ability to induce oocyte maturation, causing the resumption of the cell cycle from its arrested state. We have identified the product of a novel Xenopus gene, Speedy or Spy1, which is able to induce rapid maturation of Xenopus oocytes, resulting in the induction of germinal vesicle breakdown (GVBD) and activation of M-phasepromoting factor (MPF). Spy1 activates the MAPK pathway in oocytes, and its ability to induce maturation is dependent upon this pathway. Spy1-induced maturation occurs much more rapidly than maturation induced by other cell cycle regulators including progesterone, mos or Ras, and does not require any of these proteins or hormones, indicating that Spy1-induced maturation proceeds through a novel regulatory pathway. In addition, we have shown that Spy1 physically interacts with cdk2, and prematurely activates cdk2 kinase activity. Spy1 therefore represents a novel cell cycle regulatory protein, inducing maturation through the activation of MAPK and MPF, and also leading to the premature activation of cdk2.

Amino Acid Sequence↗
PubMed1999

Analysis of the peroxisomal acyl-CoA oxidase gene product from Pichia pastoris and determination of its targeting signal.

Acyl-CoA oxidase (Pox1p) is involved in the beta-oxidation of fatty acids and is targeted to the peroxisomal matrix via the use of different signals in various organisms. In rat, mouse and human, Pox1p contains a canonical peroxisomal targeting signal 1 (PTS1), whereas in the yeasts Candida tropicalis, Saccharomyces cerevisiae, C. maltosa and Yarrowia lipolytica neither a PTS1 nor a PTS2 sequence is present, suggesting that Pox1p might be targeted to the peroxisomes via a third unknown pathway. Alternatively, since proteins lacking a PTS sequence can enter peroxisomes in association with other polypeptides containing a PTS, Pox1p might 'piggy-back' its way into the peroxisomal matrix together with other proteins. To understand the mechanism of peroxisomal targeting of a yeast Pox1p, we cloned the Pichia pastoris POX1 gene to study the pathway of import of PpPox1p into peroxisomes. The gene was cloned by PCR, hybridization and plasmid rescue. The 2157 bp gene encodes a protein with a predicted molecular weight of 80 kDa. Antisera against PpPox1p detected a protein specifically induced on oleate with an apparent molecular weight of 72 kDa. Immunolocalization studies confirmed the peroxisomal localization of PpPox1p. The carboxy-terminus of PpPox1p ends with a PTS1-like sequence, APKI. The sequence PKI was necessary for transport of PpPox1p into peroxisomes and interacted with the PTS1 receptor, Pex5p. Furthermore, addition of the sequence APKI to the C-terminus of the green fluorescent protein directed this fusion protein to the peroxisome. Therefore, PpPox1p uses the PTS1 pathway for its import into peroxisomes.

Acyl-CoA Oxidase↗