Prenylcysteine a-Carboxyl Methyltransferase in Suspension-Cultured Tobacco Cells1 (original) (raw)
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Cysteine Metabolism in Cultured Tobacco Cells
Plant Physiology, 1980
Transported L-135Sicysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight. The 35S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal L-536SIcysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the 36S label present as compounds other than cysteine. The initial step in cysteine degradation yielded pyruvate, suffide, and presumably NH4'. Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K. to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria. The regulation of sulfate reduction and the biosynthesis of the sulfur-containing amino acids has been reviewed recently by Wilson and Reuveny (29). Cysteine or a close metabolite is thought to be an important regulatory compound in bacterial and fungal systems (2, 15, 16, 20, 27). At least one enzyme involved in sulfate reduction (ATP-sulfurylase) in tobacco cells is thought to be subject to end product repression by cysteine or a close metabolite and derepression during growth on more slowly assimilated sulfur sources (22, 23). In Lemna, adenosine 5'-phosphosulfate sulfotransferase activity is inhibited in vitro by cysteine, and sulfide pretreatment results in decreased extractable activity of this enzyme (3). These observations caused these workers to conclude that the internal cysteine and/or sulfide pool regulate the sulfur assimilation pathway in Lemna (3). Cysteine inhibits sulfate transport into cultured tobacco cells (14) and potato tubers (19). Smith (25) has demonstrated that cysteine may contribute sulfur to the sulfate pool in tobacco cells. When tobacco cells are incubated in L-[35S]cysteine, the internal cysteine pool is elevated to a "maximum level" and the excess cysteine is degraded with the sulfur being metabolized to sulfate.
THE PLANT CELL ONLINE, 2007
Cys synthesis in plants constitutes the entry of reduced sulfur from assimilatory sulfate reduction into metabolism. The catalyzing enzymes serine acetyltransferase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL) reversibly form the heterooligomeric Cys synthase complex (CSC). Dominant-negative mutation of the CSC showed the crucial function for the regulation of Cys biosynthesis in vivo. An Arabidopsis thaliana SAT was overexpressed in the cytosol of transgenic tobacco (Nicotiana tabacum) plants in either enzymatically active or inactive forms that were both shown to interact efficiently with endogenous tobacco OAS-TL proteins. Active SAT expression resulted in a 40-fold increase in SAT activity and strong increases in the reaction intermediate OAS as well as Cys, glutathione, Met, and total sulfur contents. However, inactive SAT expression produced much greater enhancing effects, including 30-fold increased Cys levels, attributable, apparently, to the competition of inactive transgenic SAT with endogenous tobacco SAT for binding to OAS-TL. Expression levels of tobacco SAT and OAS-TL remained unaffected. Flux control coefficients suggested that the accumulation of OAS and Cys in both types of transgenic plants was accomplished by different mechanisms. These data provide evidence that the CSC and its subcellular compartmentation play a crucial role in the control of Cys biosynthesis, a unique function for a plant metabolic protein complex.
THE PLANT CELL ONLINE, 2009
Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose...
Bioscience, biotechnology, and biochemistry, 2015
S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.
Cysteine Transport into Cultured Tobacco Cells
PLANT PHYSIOLOGY, 1977
Cysteine trnsport by tobacco cells (Nicodana tabacum L. var. Xanthi) cultured on liquid 5-5 medim was exaed. Transport was linear with time or mount of tissue and had a pH optimum of 4.5. Cysteine trasport over a wide concentration range was biphasic. The iotherm, for descriptive convenience, was divided into two segments both of which obeyed Michneli-Menten kinetics. The Km for high affinity ransport was in the range 1.7 x 10-5 M(±0.17) while the Kin for low affinity transport was in the range 3.5 x 104 M(±0.13). Maxinm velocities were 3 to 6 umoles/g hfesh weight/mnute and 13 to 16 umoes/g fes weight/minute, respectively. Azide ad 2,44-nitrophenol caused more than 90% inhibition of net tansport by either system. N,N'-Dicydohexykcrbodlmide was not inhitory while the inbibition by carbonylcyanide m-clorophenylhydrazone was dependent on the cysteine concentration. Only those compou,ds that were inhibitory to ransport caused efflux of labeled material from preloaded cells. Tobacco cells that had been preincubated in iodoacetamide or Nethylmaeimide did not transport cysteine while simiar treatments with dihothreitol were only slightly inhibitory or had no effect on btasport. Transport by either system was, to some extent, inhibited by nlU other tested amino acids and analogs. Alnine, methionine, and Smethyl cysteine were most effective in inhibiig cysteine rnsprt. Both alanine and methionine were competitive inhibitors of cysteine trnport by either system with inhibition constants that were imilar to the Km for the particlar system. The transport of amino acids has been studied in bacterial systems (23). Lombardi and Kaback (16) provided evidence for
Protein farnesylation in plants: a greasy tale
Current Opinion in Plant Biology - CURR OPIN PLANT BIOL, 1999
Although farnesylation is required for a number of abscisic acid mediated responses in plants, knowledge of how this lipid modification of proteins regulates specific developmental and physiological processes remains unclear. Recent information from the Arabidopsis genome-sequencing project in combination with mutants deficient in farnesylation should unravel the role(s) of this process in plant signaling.
Protein prenylation in spinach chloroplasts
Proceedings of the National Academy of Sciences of the United States of America, 1999
Protein prenylation in plants was studied by in vivo metabolic (3)H-mevalonate labeling in combination with a range of protein synthesis inhibitors. In spinach cotyledons, this posttranslational protein modification was found to be divided into two categories, one representing the conventional prenylation involving farnesyl and geranylgeranyl groups bound to cysteine residues via thioether linkages. This category revealed a similar pattern of prenylated proteins to that observed in mammalian cells and depends on nuclear gene expression. The other category was shown to represent a type of prenylation confined to chloroplasts. It depends on plastid gene expression and does not involve a thioether bond. The modifying isoprenoid could be released from the chloroplastic polypeptides by alkaline treatment and was identified as phytol upon GC-MS analysis. The phytol could readily be derived from all-trans-[(3)H]farnesol, which, like all-trans-[(3)H]geranylgeraniol, was taken up by the coty...