Km mutants of the Chlorella monosaccharide/H+ cotransporter randomly generated by PCR.

Will, Andreas; Caspari, Thomas; Tanner, Widmar

doi:10.1073/pnas.91.21.10163

Cited by 26 publications

(24 citation statements)

References 31 publications

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“…3A). In addition, an Asp residue essential for the activity of the C. kessleri HUP1 gene in Schizosaccharomyces pombe was conserved at position 39 of PMT1, as well as the residues V433 and N436 of HUP1, which compared to V428 and N431 of PMT1 (Caspari et al, 1994;Will et al, 1994). Analogous to the earlier reported transmembrane sugar transporters, PMT1 contains 12 putative transmembrane regions (Sauer and Tanner, 1993; Fig.…”

Section: Discussionmentioning

confidence: 81%

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Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

et al. 1998

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We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

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Section: Discussionmentioning

confidence: 81%

“…PMT1 contains all conserved amino acids and motifs: Q175, Q293, Q294, HWFW490-493, (R/K)-GR(R/K)343-347, and (V/ L)PETKG472-476 (Sauer and Stadler, 1993;Caspari et al, 1994;Will et al, 1994;Harrison, 1996;Fig. 3A).…”

Section: Discussionmentioning

confidence: 99%

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

et al. 1998

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“…Although there is one published report of changing the selectivity of the wheat high affinity K ϩ -Na ϩ transporter with a single amino acid substitution (21), most examples of single amino acid substitutions affecting transporter activity either altered transporter activity with respect to one or more substrates or decreased transporter selectivity (22)(23)(24)(25). The IRT1 examples discussed above demonstrate that we can alter the metal transport profile of a ZIP family member.…”

Section: Resultsmentioning

confidence: 99%

Altered selectivity in anArabidopsismetal transporter

Rogers

Eide

Guerinot

2000

Proc. Natl. Acad. Sci. U.S.A.

432

322

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Plants require metals for essential functions ranging from respiration to photosynthesis. These metals also contribute to the nutritional value of plants for both humans and livestock. Additionally, plants have the ability to accumulate nonessential metals such as cadmium and lead, and this ability could be harnessed to remove pollutant metals from the environment. Designing a transporter that specifically accumulates certain cations while excluding others has exciting applications in all of these areas. The Arabidopsis root membrane protein IRT1 is likely to be responsible for uptake of iron from the soil. Like other Fe(II) transporters identified to date, IRT1 transports a variety of other cations, including the essential metals zinc and manganese as well as the toxic metal cadmium. By heterologous expression in yeast, we show here that the replacement of a glutamic acid residue at position 103 in wild-type IRT1 with alanine increases the substrate specificity of the transporter by selectively eliminating its ability to transport zinc. Two other mutations, replacing the aspartic acid residues at either positions 100 or 136 with alanine, also increase IRT1 metal selectivity by eliminating transport of both iron and manganese. A number of other conserved residues in or near transmembrane domains appear to be essential for all transport function. Therefore, this study identifies at least some of the residues important for substrate selection and transport in a protein belonging to the ZIP gene family, a large transporter family found in a wide variety of organisms. T he primary control point for metal ion homeostasis appears to be regulation of metal uptake across the plasma membrane. One important question that arises is whether there are separate transporters for each metal and, if not, which metals share the same transporter system. We have recently identified a group of metal transporters, the ZIP gene family, whose members have been implicated in the transport of a variety of metals (1). IRT1, the first ZIP gene to be identified, was discovered by functional complementation of the fet3 fet4 iron uptake mutant of Saccharomyces cerevisiae (2). IRT1 is also capable of complementing the metal uptake defects of the S. cerevisiae zrt1 zrt2 zinc uptake mutant and the S. cerevisiae smf1 manganese uptake mutant (3). This IRT1 complementation data, along with results from radioactive uptake assays (2, 3), demonstrate that IRT1 is capable of transporting Fe, Zn, and Mn; sensitivity and accumulation studies show that IRT1 can also transport cadmium (E. L. Connolly and M.L.G., unpublished data and this work). In this respect, IRT1 is similar to other iron transporters described to date (4, 5).The broad cation range of IRT1 makes it an excellent model system for the study of residues involved metal recognition and transport. To determine the importance of individual amino acid side chains for metal transport, we substituted alanine for a number of amino acid residues that are highly conserved among ZIP family members and ...

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“…TIe limited data which are currently available have been obtained mainly using site-directed mutagenesis (especially with th~: E. coli lactose permease: [6,7]) or random mutagenesis (with the: Chlorella kessleri HUP1 glucose-H + symporter [8] A~breviations: CCCP, carbonyl cyanide m-chlorophenyl hydrazone; cy,,ochrome-e-oxidase, ferrocytochrome e:oxygen oxidoreductase (EC 1.9.3.1); OG, octyl-fl-D-glucoside; TMPD, N,N,N',N'-tetramethyl-pph enylenediamine.…”

Section: Introductionmentioning

confidence: 99%

Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain

1995

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The only way to obtain a clear picture of the structure of these proteins is the generation of 2-dimensional or 3-dimensional crystals or the structural analysis of purified protein by NMR. For most membrane proteins, however, already the first steps towards any of these techniques are hindered by the low expression levels of these proteins, by their extreme hydrophobicity and the concomitant difficulties during purification.The attachment of hydrophilic tags to such proteins has become an important tool to overcome at least part of these problems [9 12]. The present paper describes the advantages of a bacterial biotin acceptor domain for the one-step purification of the recombinant P major sucrose carrier PmSUC2 [13] from transgenic yeast.

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Km mutants of the Chlorella monosaccharide/H+ cotransporter randomly generated by PCR.

Cited by 26 publications

References 31 publications

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

Altered selectivity in anArabidopsismetal transporter

Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain

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