Abstracts of papers (2001)

Last Update: 09/07/2001

Abstracts of papers (2001)

[2001-1] Tomitori, H. et al., Biolchem. J. 353, 681-688 (2001)

We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast [Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J. Biol. Chem. 274, 3265-3267]. Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A1, an inhibitor of the vacuolar H+-ATPase. Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles. Furthermore, it was shown that Glu-207, Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important for the transport activity.

[2001-2] Kawano, M. et al., Arch. Microbiol. 175, 41-45 (2001)

Two high-affinity K+ uptake systems, KtrI and KtrII, have been reported in Enterococcus hirae. A mutant, JEMK1, defective in these two systems did not grow at pH 10 in low-K+ medium (less than 1 mM K+), but grew well when supplemented with 10 mM KCl. In this mutant, we found an energy-dependent K+ uptake at pH 10 with a low affinity for K+ (Km of ~20 mM) and an extremely high rate [Vmax of 1.6 オmol min-1 (mg protein)-1]. Rb+ uptake [Km of ~40 mM and Vmax of 0.5 オmol min-1 (mg protein)-1], which was inhibited competitively by K+ and less prominently by Cs+, was also observed. The specificity of this transport is likely to be K+>Rb+>Cs+. This peculiar K+ transport plays a role as a salvage mechanism against defects in high-affinity systems in the K+ homeostasis of this bacterium.

[2001-3] Nitta, T. et al., Exp. Cell Res. 265, 174-183 (2001)

The B cell lymphoma WEHI231 has been used as a model for studying clonal deletion of B cells on the basis of its ability to undergo growth arrest and apoptosis by B cell antigen receptor (BCR) cross-linking. To comprehensively analyze the genes involved in BCR-mediated apoptosis, we applied the technique of serial analysis of gene expression (SAGE) to WEHI231. Comparison of expression patterns revealed that BCR cross-linking caused coordinate changes in the expression of genes involved in polyamine metabolism. Polyamines are ubiquitous compounds required for cell proliferation and homeostasis. The coordinate expression of the polyamine-related genes was confirmed by semiquantitative reverse transcriptase-polymerase chain reaction analysis. During apoptosis, the genes involved in polyamine biosynthesis were downregulated, whereas those involved in polyamine catabolism were upregulated, suggesting that intracellular polyamines play a role in BCR-mediated apoptosis. Levels of intracellular putrescine, spermidine, and spermine were reduced after BCR cross-linking. These effects were prevented by concurrent CD40 stimulation, which blocked BCR-mediated apoptosis. Furthermore, addition of spermine could repress the BCR-mediated apoptosis by attenuating the mitochondrial membrane potential (Dym) loss and activation of caspase-7 induced by BCR signaling. These findings strongly suggest that polyamine regulation is involved in apoptosis during B cell clonal deletion.

[2001-4] Sharmin, S. et al., Biochem. Biophys. Res. Commun. 282, 228-235 (2001)

The toxicity of extracellular spermine, determined in the presence of fetal calf serum, was studied using three cell lines: FM3A, L1210, and NIH3T3 cells. Amine oxidase in fetal calf serum produces aminodialdehyde generating acrolein spontaneously, H2O2, and ammonia from spermine. Spermine toxicity was prevented by aldehyde dehydrogenase, but not by catalase. Similar concentrations of spermine and acrolein were needed to produce toxicity. Other aldehydes (formaldehyde, acetaldehyde, and propionaldehyde) and hydrogen peroxide were less toxic than acrolein. Spermidine and 3-aminopropanal, which produces acrolein, also exhibited severe cytotoxicity. The degree of cytotoxicity of spermine, spermidine, and 3-aminopropanal was nearly parallel with the amount of acrolein produced from each compound. Thus, it was deduced that acrolein is a major toxic compound produced from polyamines (spermine and spermidine) by amine oxidase.

[2001-5] Murata, T. et al., Biochim. BIOphys. Acta 1505, 75-81 (2001)

V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na(+)-translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na(+)-ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K(+) transporter independent of the Na(+)-ATPase. Expression of this operon, encoding two transport systems for Na(+) and K(+) ions, was regulated at the transcriptional level by intracellular Na(+) as the signal. Structural aspects and catalytic properties of purified Na(+)-ATPase closely resembled those of other V-type H(+)-ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na(+) at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na(+) binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na(+) binding sites of the enzyme.

[2001-6] Yoshida, M. et al., J. Biol. Chem. 276, 16289-16295 (2001)

The effects of polyamines on the synthesis of various sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of sigma28 was stimulated 4.0-fold and that of sigma38 was stimulated 2.3-fold by polyamines, whereas synthesis of other sigma subunits was not influenced by polyamines. Stimulation of sigma28 synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.

[2001-7] Igarashi, K. et al., Res. Microbiol. 152, 271-278 (2001)

The polyamine content of cells is regulated by biosynthesis, degradation, and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) are ABC transporters, each consisting of a periplasmic substrate binding protein, two transmembrane proteins, and a membrane-associated ATPase. The third transport system, catalyzed by PotE, mediates both uptake and excretion of putrescine. In this article, the properties of the first two polyamine uptake systems are reviewed in detail.

[2001-8] Raj, V. S. et al., J. Bacteriol. 183, 4493-4498 (2001)

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma38 subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduced the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.