Control of Free Methionine Production in Wild Type and Ethionine-resistant Mutants of <i>Chlorella sorokiniana</i>

Sloger, Marcia; Owens, Lowell D.

doi:10.1104/pp.53.3.469

Cited by 14 publications

(5 citation statements)

References 17 publications

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“…Individual amino acids were identified by decending paper chromatography on Whatman No. 3MM paper, using methanol-pyridine-1.25 N HCI (37:4:8) as solvent (29). RESULTS (9).…”

Section: Methodsmentioning

confidence: 99%

Sulfate Transport in Cultured Tobacco Cells

Smith¹

1975

Plant Physiol.

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In Neurospora crassa, evidence has been presented for two distinct sulfate permeases coded for by two unlinked genes (12)(13)(14)26). One of the permeases predominates in the conidial stage and the other predominates during mycelial growth (13). The transport system is energy-dependent, highly temperature-dependent, repressed by high methionine, and subject to positive control by the product of the cys-3 locus (15). Other filamentous fungi also contain a metabolically controlled sulfate permease system that is repressed by Lmethionine, and feedback inhibited by intracellular sulfate and possibly cysteine or a cysteine metabolite (1, 32).Sulfate transport in higher plants has been investigated using either plant roots (6,11,16,23,24) or plant cells cultured in liquid media (9). In cultured tobacco cells, there seems to be a negative feedback control of sulfate assimilation by methionine and cysteine (9), similar to that previously reported in microorganisms.Although Pardee and coworkers (17-21) presented evidence to support protein-mediated active transport of sulfate in bacteria, most of the work with eukaryotes is based on indirect evidence. Studies were initiated to obtain direct evidence for the presence of a sulfate permease system in cultured tobacco cells. Preliminary investigations indicated that the size of the intracellular sulfate pool might be a major factor in controlling the rate of sulfate transport in these cells. MATERIALS AND METHODSTobacco XD-cell line (Nicotiana tabacum L. var. Xanthi) was obtained from P. Filner and cultured as previously described (9). Sulfateless M-1D medium was prepared, and sulfate (50 ,M, final concentration) was added before sterilization, or L-cysteine (50 juM, final concentration) was added through a Millipore G.S. filter following medium sterilization.TranWort Experiments. Cells were harvested by vacuum filtration and washed with 100 ml of medium M-1D (minus sulfate). The washed cells (0.25-0.5 g fresh weight) were placed in a 50-ml Erlenmeyer flask containing 20 ml of M-1D medium and 0.2 ml of 5 mm Na2 "SOI (1 MCi). The flasks were stoppered with cotton plugs and placed on a rotary shaker at 25 C. At the termination of the transport experiment, cells were harvested by vacuum filtration and washed with 0.5 mm sodium sulfate (100 ml) to remove adsorbed radioactive sulfate. Transported sulfate was extracted by placing cells in 20 ml of boiling H,O for 1 min. A 5-ml aliquot of this extract was added to 10 ml of Aquasol liquid scintillation fluid and was counted in a Packard 3310 Tri-Carb scintillation spectrometer with external standardization.In preincubation experiments, cells were washed as decribed above, suspended in 20 ml of preincubation medium in an Erlenmeyer flask, and shaken on a rotary shaker at 25 C. Following preincubation, cells were harvested and washed with 100 ml of M-ID medium (minus sulfate) before the measurement of transport.All operations before the addition of radioactive sulfate were conducted in a sterile room using sterilized media and equipme...

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“…Individual amino acids were identified by decending paper chromatography on Whatman No. 3MM paper, using methanol-pyridine-1.25 N HCI (37:4:8) as solvent (29). RESULTS (9).…”

Section: Methodsmentioning

confidence: 99%

Sulfate Transport in Cultured Tobacco Cells

Smith¹

1975

Plant Physiol.

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“…The toxicity of ethionine is explained by the fact that ethionine is used in place of Met in cellular processes such as protein synthesis and SAM synthesis (Alix, 1982). Mutants that are resistant to ethionine have been reported in severa1 organisms, including prokaryotes (Lawrence et al, 1968;Greene et al, 1970), lower eukaryotes (Spence et al, 1967;Cherest et al, 1973;Sloger and Owens, 1974), and cultured plant cells (Widholm, 1976;Reish et al, 1981;Gonzales et al, 1984;Madison and Thompson, 1988). These ethionine-resistant mutants are reported to overaccumulate soluble Met.…”

Section: ~~mentioning

confidence: 99%

Isolation of an Arabidopsis thaliana Mutant, mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation)

Inaba

Fujiwara

Hayashi³

et al. 1994

Plant Physiol.

142

101

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“…One mechanism of resistance would be to alter metabolic regulation, such that an overproduction of one or more natural amino acids could effectively counteract the deleterious effects of the selec tive agent(s). Over production of methionine has been observed in cell lines of carrots (Widholm, 1976) and chlorella (Sloger and Owens, 1974) after selection for resistance to ethionine. Tobacco cell lines resistant to δ-hydroxylysine orS-2-aminoethyl-L-cysteine (AEC) were characterized by up to 10-fold increases in free lysine, and found to be resistant for up to 70 cell doublings in the absence of the selective agent (Widholm, 1976).…”

Section: A Mutant Analysismentioning

confidence: 99%