Multiple polyamine transport systems on the vacuolar membrane in yeast

2001 ◽  
Vol 353 (3) ◽  
pp. 681-688 ◽  
Author(s):  
Hideyuki TOMITORI ◽  
Keiko KASHIWAGI ◽  
Tomoko ASAKAWA ◽  
Yoshimi KAKINUMA ◽  
Anthony J. MICHAEL ◽  
...  
Keyword(s):  
Transport Protein ◽  
Vacuolar Membrane ◽  
Open Reading Frames ◽  
Transport Systems ◽  
Polyamine Transport ◽  
Polyamine Content ◽  
Increased Sensitivity ◽  
Uptake Activity ◽  
Polyamine Uptake ◽  
Reading Frames

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.

scholarly journals Identification of an ATP-Driven, Osmoregulated Glycine Betaine Transport System in Listeria monocytogenes

1999 ◽  
Vol 65 (9) ◽  
pp. 4040-4048 ◽  
Author(s):  
Rinkei Ko ◽  
Linda Tombras Smith
Keyword(s):  
Listeria Monocytogenes ◽  
Glycine Betaine ◽  
Open Reading Frames ◽  
Transport Systems ◽  
High Concentration ◽  
Promoter Regions ◽  
Reading Frame ◽  
Transposon Insertion ◽  
Food Borne ◽  
Reading Frames

ABSTRACT The ability of the gram-positive, food-borne pathogenListeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na+-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na+ for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuAand proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na+ is an indication that only the ATP-dependent transporter and the Na+-glycine betaine symporter occur in L. monocytogenes.

scholarly journals Whole-Genome Analysis of Transporters in the Plant Pathogen Xylella fastidiosa

2002 ◽  
Vol 66 (2) ◽  
pp. 272-299 ◽  
Author(s):  
Joao Meidanis ◽  
Marilia D. V. Braga ◽  
Sergio Verjovski-Almeida
Keyword(s):  
Xylella Fastidiosa ◽  
Open Reading Frames ◽  
Transport Systems ◽  
Phosphotransferase System ◽  
Whole Genome Analysis ◽  
Plant Parasite ◽  
Intracellular Parasites ◽  
P Type ◽  
Import System ◽  
Reading Frames

SUMMARY The transport systems of the first completely sequenced genome of a plant parasite, Xylella fastidiosa, were analyzed. In all, 209 proteins were classified here as constitutive members of transport families; thus, we have identified 69 new transporters in addition to the 140 previously annotated. The analysis lead to several hints on potential ways of controlling the disease it causes on citrus trees. An ADP:ATP translocator, previously found in intracellular parasites only, was found in X. fastidiosa. A P-type ATPase is missing—among the 24 completely sequenced eubacteria to date, only three (including X. fastidiosa) do not have a P-type ATPase, and they are all parasites transmitted by insect vectors. An incomplete phosphotransferase system (PTS) was found, without the permease subunits—we conjecture either that they are among the hypothetical proteins or that the PTS plays a solely metabolic regulatory role. We propose that the Ttg2 ABC system might be an import system eventually involved in glutamate import rather than a toluene exporter, as previously annotated. X. fastidiosa exhibits fewer proteins with ≥4 α-helical transmembrane spanners than any other completely sequenced prokaryote to date. X. fastidiosa has only 2.7% of all open reading frames identifiable as major transporters, which puts it as the eubacterium having the lowest percentage of open reading frames involved in transport, closer to two archaea, Methanococcus jannaschii (2.4%) and Methanobacterium thermoautotrophicum (2.4%).

scholarly journals Study of the Five Rickettsia prowazekii Proteins Annotated as ATP/ADP Translocases (Tlc): Only Tlc1 Transports ATP/ADP, While Tlc4 and Tlc5 Transport Other Ribonucleotides

2006 ◽  
Vol 188 (17) ◽  
pp. 6261-6268 ◽  
Author(s):  
Jonathon P. Audia ◽  
Herbert H. Winkler
Keyword(s):  
Heterologous Expression ◽  
Host Cell ◽  
De Novo ◽  
Open Reading Frames ◽  
Transport Systems ◽  
Heterologous Protein Expression ◽  
Rickettsia Prowazekii ◽  
E Coli ◽  
Nucleic Acid Biosynthesis ◽  
Reading Frames

ABSTRACT The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a Ki similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.

scholarly journals Multiple polyamine transport systems on the vacuolar membrane in yeast

2001 ◽  
Vol 353 (3) ◽  
pp. 681 ◽  
Author(s):  
Hideyuki TOMITORI ◽  
Keiko KASHIWAGI ◽  
Tomoko ASAKAWA ◽  
Yoshimi KAKINUMA ◽  
Anthony J. MICHAEL ◽  
...  
Keyword(s):  
Vacuolar Membrane ◽  
Transport Systems ◽  
Polyamine Transport

scholarly journals Polyamine transport in bacteria and yeast

1999 ◽  
Vol 344 (3) ◽  
pp. 633-642 ◽  
Author(s):  
Kazuei IGARASHI ◽  
Keiko KASHIWAGI
Keyword(s):  
Binding Proteins ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Transport Systems ◽  
Uptake System ◽  
Polyamine Transport ◽  
Polyamine Content ◽  
Methylene Groups ◽  
Preferential Uptake

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) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating β-α-β topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH2- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.

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