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. 1998 Sep;118(1):115-23.
doi: 10.1104/pp.118.1.115.

Prenylcysteine alpha-carboxyl methyltransferase in suspension-cultured tobacco cells

DN Crowell  1 , SE Sen, SK Randall
Affiliations

Prenylcysteine alpha-carboxyl methyltransferase in suspension-cultured tobacco cells

DN Crowell et al. Plant Physiol. 1998 Sep.

Abstract

Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes. Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells. Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described. Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine alpha-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 &mgr;M for N-acetyl-S-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, respectively. Furthermore, competition analysis indicates that the same enzyme is responsible for both activities. These results suggest that alpha-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants.

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Figures

Figure 1
Figure 1
Reaction mechanism of prenylcysteine α-carboxyl methyltransferase (Shi and Rando, 1992).
Figure 2
Figure 2
Farnesylcysteine and geranylgeranylcysteine α-carboxyl methyltransferase assays on isolated membranes from cultured tobacco BY-2 cells. Assays were performed essentially as described by Hrycyna and Clarke (1990). Production of base-labile radioactivity was measured as a function of time in the presence of tobacco membranes, S-adenosyl-l-[3H-methyl]Met, and 200 μm AFC, AGGC, or AGC. The background in the absence of exogenous methyl acceptor was identical to that detected in the presence of 200 μm AGC (data not shown).
Figure 3
Figure 3
Modified assay for tobacco farnesylcysteine and geranylgeranylcysteine α-carboxyl methyltransferase. Assays were performed in the presence of tobacco membranes, S-adenosyl-l-[3H-methyl]Met, and 200 μm AFC, AGGC, AGC, or no exogenous methyl acceptor. Assay mixtures were then resolved by silica gel TLC either before (A) or after (B) extraction into 90% methylene chloride, 9.75% methanol, and 0.25% acetic acid.
Figure 4
Figure 4
pH optimum for tobacco prenylcysteine α-carboxyl methyltransferase. Assays were performed in the presence of 200 μm AGGC or AGC using the following buffers at 100 mm: sodium acetate at pH 5.48, sodium acetate at pH 5.97, sodium acetate at pH 6.46, Hepes at pH 6.76, Hepes at pH 7.00, and Hepes at pH 7.61.
Figure 5
Figure 5
Time-dependent farnesylcysteine and geranylgeranylcysteine α-carboxyl methyltransferase activities. Product formation in the presence of 200 μm AFC, AGGC, or AGC was observed over a 60-min period at 30°C in 0.029 mg of tobacco membrane protein. Arrows indicate the positions of relevant methylated products and the origins at which samples were spotted before silica gel TLC. The plot shown was generated by subtracting the AGC background.
Figure 6
Figure 6
Protein-dependent farnesylcysteine and geranylgeranylcysteine α-carboxyl methyltransferase activities. Product formation in the presence of 200 μm AFC, AGGC, or AGC was measured as a function of the amount of tobacco membrane protein in the 60-min assay at 30°C. Arrows indicate the positions of relevant methylated products and the origins at which samples were spotted before silica gel TLC. The plot shown was generated by subtracting the AGC background. The background is less obvious than in Figures 3–5 because the fluorograms shown represent relatively short exposures.
Figure 7
Figure 7
Comigration of reaction products with chemically synthesized AFC and AGGC methyl esters by HPLC. HPLC elution profiles are shown for tritiated reaction products generated in the presence of AFC (top) or AGGC (bottom). The positions of AFC, AFC methyl ester (AFC Me), AGGC, and AGGC methyl ester (AGGC Me) standards were determined by A214 and are indicated by arrows.
Figure 8
Figure 8
Kinetic analyses of tobacco farnesylcysteine and geranylgeranylcysteine α-carboxyl methyltransferase activities. Activity curves are shown as a function of AFC (top) or AGGC (bottom) concentration. The plot shown was generated by subtracting the AGC background. Km and Vmax values were determined by Lineweaver-Burk analysis of the data (not shown).
Figure 9
Figure 9
Competition analyses suggest that AFC and AGGC are α-carboxyl methylated by the same enzyme. Prenylcysteine α-carboxyl methyltransferase assays were performed in the presence of AFC, AGGC, or both at the concentrations indicated below the graph. In A, AFC was used at its apparent Km and AGGC at five times its apparent Km. In B, AFC was used at five times its apparent Km and AGGC at its apparent Km. Samples were analyzed by quantitative HPLC. The black bars represent AFC α-carboxyl methylation and the striped bars represent AGGC α-carboxyl methylation.
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References

    1. Beranger F, Cadwallader K, Porfiri E, Powers S, Evans T, de Gunzberg J, Hancock JF. Determination of structural requirements for the interaction of Rab6 with RabGDI and Rab geranylgeranyltransferase. J Biol Chem. 1994;269:13637–13643. - PubMed
    1. Biermann BJ, Morehead TA, Tate SE, Price JR, Randall SK, Crowell DN. Novel isoprenylated proteins identified by an expression library screen. J Biol Chem. 1994;269:25251–25254. - PubMed
    1. Boyartchuk VL, Ashby MN, Rine J. Modulation of Ras and a-factor function by carboxyl-terminal proteolysis. Science. 1997;275:1796–1800. - PubMed
    1. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. - PubMed
    1. Clarke S, Vogel JP, Deschenes RJ, Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci USA. 1988;85:4643–4647. - PMC - PubMed

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