Yoshimi Kakinuma

Multiple polyamine transport systems on the vacuolar membrane in yeast

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 A(1), 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.

Identification of a gene for a polyamine transport protein in yeast.

Properties of a membrane protein encoded byYLL028w were examined using yeast cells transformed with the gene. The transformed cells became resistant to polyamine toxicity, and the resistance was overcome by bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Although spermine uptake activity of the transformed cells was almost the same as that of wild type cells, the uptake activity of vacuolar membrane vesicles from the transformed cells was higher than that from wild type cells. The transformed cells became resistant to MGBG (methylglyoxal bis(guanylhydrazone)) and paraquat, but not Ni2+ and Co2+, suggesting that the protein encoded byYLL028w is a transport protein specific for polyamines. When the YLL028w gene was disrupted by inserting theHIS3 gene, the cells became sensitive to polyamines, and spermine uptake activity of the vacuolar membrane vesicles decreased significantly. The accumulated spermine in YLL028wgene-disrupted cells decreased greatly compared with that in wild type cells. The results indicate that a membrane protein encoded byYLL028w (TPO1) is a polyamine transport protein on the vacuolar membrane.

Purification and Reconstitution of Na+-translocating Vacuolar ATPase from Enterococcus hirae

Vacuolar ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. An unusual member of this family, a sodium-translocating ATPase, has been found in the eubacterium Enterococcus hirae. We report here the purification of enterococcal Na+-ATPase from the plasma membrane of cells, whose ATPase content was highly amplified by expression of the cloned ntp operon that encodes this Na+-ATPase (ntpFIKECGABDHJ). The purified enzyme appears to consist of nine Ntp polypeptides, all the above except for the ntpH and ntpJ gene products. ATPase activity was strictly dependent on the presence of Na+ or Li+ ions and was inhibited by nitrate,N-ethylmaleimide, and the peptide antibiotic destruxin B. When the purified ATPase was reconstituted into liposomes prepared fromEnterococcus faecalis phospholipids, ATP-driven Na+ uptake was observed; uptake was blocked by nitrate, destruxin B, and monensin, but it accelerated by carbonyl cyanidem-chlorophenylhydrazone and valinomycin. These data demonstrate that E. hirae Na+-ATPase is an electrogenic sodium pump of the vacuolar type. This is a promising system for research on the fundamental molecular structure and mechanism of vacuolar ATPase.

Some features of the Streptococcus faecalis Na+‐ATPase resemble thoseof the vacuolar‐type ATPases

In the ethylenediaminetetraacetic acid (EDTA) extract prepared from the membranes of Streptococcus faecalis, we found the 330‐kDa protein that was coordinately increased with the induction of Na+‐ATPase. It was missed in the EDTA extract of Nakl, a mutant defective in the Na+‐ATPase, but restored in that of its revertant, NaklR. The 330‐kDa protein showed the ATP hydrolytic activity by active staining, and mainly consisted of the polypeptides of 73 kDa, 52 kDa and possibly 38 kDa. In addition, the Na+‐stimulated ATPase of the membranes was sensitive to both nitrate and N‐ethylmaleimide, inhibitors for the vacuolar H+‐ATPase. Thus, the Na+‐ATPase of this organism has a structure similar to vacuolar H+‐ATPase.

Structure and function of vacuolar Na+-translocating ATPase in Enterococcus hirae.

A Na+-translocating ATPase was discovered in agram-positive bacterium Enterococcus hirae. Our biochemical andmolecular biological studies revealed that this Na+-ATPasebelongs to the vacuolar-type enzyme. Purified Na+-ATPaseconsisted of nine subunits: NtpA, B, C, D, E, F, G, I, and K; reconstitutedproteoliposomes showed ATP-driven electrogenic Na+translocation. All these subunits were encoded by the ntp operon:ntpFIKECGABDHJ. The deduced amino acid sequences of the majorsubunits, A, B, and K (16 kDa proteolipid), were highly similar to those ofA, B, and proteolipid subunits of vacuolar ATPases, although the similaritiesof other subunits were moderate. The ntpJ gene encoded aK+ transporter independent of the Na+-ATPase.Expression of this operon, encoding two transport systems forNa+ and K+ ions, was regulated attranscriptional level by intracellular Na+ as the signal. Tworelated cation pumps, vacuolar Na+-ATPase andF0F1, H+-ATPase, coexist in thisbacterium.

Primary structure of the alpha-subunit of vacuolar-type Na(+)-ATPase in Enterococcus hirae. Amplification of a 1000-bp fragment by polymerase chain reaction.

A 1000‐bp fragment ofEnterococcus hirae genomic DNA was amplified by the polymerase chain reaction method, using the oligonucleotide primers designed from amino acid sequences of both amino‐terminal and a tryptic fragment of the Na+‐ATPase α‐subunit in this organism. DNA sequencing of this product revealed that the amino acid sequence of Na+‐ATPase α‐subunit is highly homologous to the corresponding sequences of large (α) subunits of vacuolar (archaebacterial) type H+‐ATPases, supporting our proposal [Kakinuma, Y. and Igarashi, K. (1990) FEBS Lett. 271, 97–101] that the Na+‐ATPase of this organism belongs to the vacuolar‐type ATPase.

Polyamine-sensitive magnesium transport in Saccharomyces cerevisiae

In Saccharomyces cerevisiae we found a toxic effect of polyamines, well-known metabolites important for cell proliferation; in magnesium-limited (50 microM Mg2+) synthetic medium, cell growth was severely inhibited by spermine, spermidine and putrescine in descending order. In conjunction with a decrease in the growth rate by the addition of 0.5 mM spermine, the internal Mg2+ content decreased and the spermine content increased. When cell growth ceased, the Mg2+ content had finally decreased to about 40% of the value before the addition of spermine (120-130 nmol/mg dry weight), and the spermine content concomitantly increased 30-fold (from 1 to 30 nmol/mg dry weight); spermine4+ apparently took the internal place of Mg2+ with a probable stoichiometry of 1:2. However, the total amount of Mg2+ retained in the cells remained constant even with the addition of spermine, suggesting that spermine blocks Mg2+ accumulation. In high (2 mM) Mg2+ medium, cell growth was hardly affected by polyamines, and an exchange of spermine and Mg2+ was minimal. Energy-dependent Mg2+ uptake by whole cells was inhibited by spermine, spermidine and putrescine in a similar manner as the growth rates. On the other hand, Mg2+ inhibited spermine uptake. These results suggest that competition takes place between extracellular spermine and Mg2+ for their accumulations. It is thus clear that polyamine-sensitive Mg2+ transport system is indispensable for the physiology of this organism.

Relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase induction, and DNA synthesis

The relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis was examined with bovine small lymphocytes stimulated by concanavalin A (Con A). The Na+/H+ antiport activity was activated immediately after addition of concanavalin A; the maximum was reached 1 h after Con A addition and the activation continued at least 6 h. With increasing concanavalin A concentrations, the activities of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis increased in a parallel manner. In the presence of HCO3- in the medium, the internal alkalinization of lymphocytes was not induced by Con A. Ornithine decarboxylase and DNA synthetic activities were not inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific inhibitor of the Na+/H+ antiporter. In contrast, in the absence of HCO3- in the medium, the internal pH was alkalinized approximately 0.06 pH units by Con A. EIPA did inhibit the alkalinization of the internal pH or DNA synthesis significantly. Ornithine decarboxylase activity was not inhibited by EIPA. These results indicate that the activation of a Na+/H+ antiporter is not a trigger for cell proliferation, but its activation is important probably through the maintenance of the internal pH optimum, especially in HCO3(-)-free medium.

Evidence for Na(+) influx via the NtpJ protein of the KtrII K(+) uptake system in Enterococcus hirae.

The ntpJ gene, a cistron located at the tail end of the vacuolar-type Na(+)-ATPase (ntp) operon of Enterococcus hirae, encodes a transporter of the KtrII K(+) uptake system. We found that K(+) accumulation in the ntpJ-disrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10. Studies of the membrane potential (DeltaPsi; inside negative) by 3, 3-dihexyloxacarbocyanine iodide fluorescence revealed that the DeltaPsi was hyperpolarized at pH 10 in JEM2; the DeltaPsi values of the parent strain ATCC 9790 and JEM2, estimated by determining the equilibrium distribution of K(+) or Rb(+) in the presence of valinomycin, were -118 and -160 mV, respectively. DeltaPsi generation at pH 10 was accomplished by an electrogenic Na(+) efflux via the Na(+)-ATPase, whose levels in the two strains were quite similar. Na(+) uptake driven by an artificially imposed DeltaPsi (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na(+) movement in addition to K(+) movement. Finally, the growth of JEM2 arrested in K(+)-limited high-Na(+) medium at pH 10 was restored by addition of valinomycin. These results suggest that NtpJ mediates electrogenic transport of K(+) as well as Na(+), that it likely mediates K(+) and Na(+) cotransport, and that Na(+) movement via NtpJ is the major Na(+) reentry pathway at high pH values.

Amplification of the Na+-ATPase of Streptococcus faecalis at alkaline pH

The Na+‐ATPase activity of Streptococcus faecalis was influenced by the medium pH. Activities of the protonophore‐resistant Na+ extrusion and the KtrII (active K+ uptake by the Na+‐ATPase) were maximal in the cells grown at pH 9.5, and were minimal in those grown at pH 6.O. In the cells grown at pH 7.5, they were moderately observed. The Na+‐stimulated ATPase activity of the cells grown at pH 9.5 was about 4‐fold higher than that of the cells grown at pH 6.O. Thus, amplification of the Na+‐ATPase is remarkable at alkaline pH in this organism, possibly by an increase of the cytoplasmic Na+ level as a signal.