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Farnesol Is Utilized for Isoprenoid Biosynthesis in Plant Cells Via Farnesyl Pyrophosphate Formed by Successive Monophosphorylation Reactions

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Farnesol Is Utilized for Isoprenoid Biosynthesis in Plant Cells Via Farnesyl Pyrophosphate Formed by Successive Monophosphorylation Reactions Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions Long Thai*, Jeffrey S. Rush*, Jude E. Maul†, Timothy Devarenne†, Dana L. Rodgers*, Joseph Chappell†‡, and Charles J. Waechter*‡ *Department of Biochemistry, Medical Center, College of Medicine, University of Kentucky, Lexington, KY 40536; and †Agronomy Department, College of Agriculture, University of Kentucky, Lexington, KY 40546-0091 Edited by Rodney B. Croteau, Washington State University, Pullman, WA and approved September 23, 1999 (received for review August 17, 1999) The ability of Nicotiana tabacum cell cultures to utilize farnesol investigations implicated F-OH as the putative nonsterol regu- (F-OH) for sterol and sesquiterpene biosynthesis was investigated. lator (12, 13). Exogenous F-OH also has been shown to affect [3H]F-OH was readily incorporated into sterols by rapidly growing several other physiological processes including: inhibition of cell cultures. However, the incorporation rate into sterols was phosphatidylcholine biosynthesis (14), progression through the reduced by greater than 70% in elicitor-treated cell cultures cell cycle (15), and induction of biochemical changes associated whereas a substantial proportion of the radioactivity was redi- with an apoptotic program (16). The recent discovery of a rected into capsidiol, an extracellular sesquiterpene phytoalexin. nuclear farnesol receptor, FXR, is consistent with a regulatory The incorporation of [3H]F-OH into sterols was inhibited by squal- role for F-OH in controlling diverse metabolic processes (17). estatin 1, suggesting that [3H]F-OH was incorporated via farnesyl Work in our laboratories has focused on the metabolic shifts pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum that occur between the central isoprenoid pathway and sterol, proteins were metabolically labeled with [3H]F-OH or [3H]gera- sesquiterpene, and dolichol biosynthesis (6, 18, 19). Although we nylgeraniol ([3H]GG-OH). Kinase activities converting F-OH to far- have measured changes in the activities of several branch point nesyl monophosphate (F-P) and, subsequently, F-P-P were demon- enzymes in vitro (6), a limitation of our work has been the strated directly by in vitro enzymatic studies. [3H]F-P and [3H]F-P-P absence of experimentation probing the in vivo functioning of were synthesized when exogenous [3H]F-OH was incubated with the downstream and branch-point portions of the isoprenoid microsomal fractions and CTP. The kinetics of formation suggested pathway. Work more than 10 years earlier provided an experi- a precursor–product relationship between [3H]F-P and [3H]F-P-P. In mental means to overcoming this limitation. In 1988 and 1989, agreement with this kinetic pattern of labeling, [32P]F-P and [32P]F- Poulter and coworkers demonstrated the incorporation of ra- P-P were synthesized when microsomal fractions were incubated diolabeled prenyl alcohols into the geranylgeranyl lipids of with F-OH and F-P, respectively, with [␥-32P]CTP serving as the Methanospirillum hungatei (20), as well as into the triterpenes of phosphoryl donor. Under similar conditions, the microsomal frac- Botryococcus braunii (21). These investigators suggested that the tions catalyzed the enzymatic conversion of [3H]GG-OH to prenol alcohols could be ‘‘activated’’ via the action of a prenol- [3H]geranylgeranyl monophosphate and [3H]geranylgeranyl pyro- kinase. In agreement with these observations, F-OH and phosphate ([3H]GG-P-P) in CTP-dependent reactions. A novel bio- GG-OH were shown to be utilized for sterol biosynthesis and synthetic mechanism involving two successive monophosphoryla- protein isoprenylation in mammalian cells (22–24). Related tion reactions was supported by the observation that [3H]CTP was studies have reported preliminary evidence for isoprenol kinases formed when microsomes were incubated with [3H]CDP and either in rat liver (25, 26), a microgreen algae (27), and an archebac- F-P-P or GG-P-P, but not F-P. These results document the presence terium (28). The current work was undertaken to determine of at least two CTP-mediated kinases that provide a mechanism for whether plant cells could utilize exogenously supplied prenol the utilization of F-OH and GG-OH for the biosynthesis of isopre- alcohols for the synthesis of isoprenoid products and, if so, to noid lipids and protein isoprenylation. determine whether the isoprenols were ‘‘activated’’ by novel pyrophosphorylation or successive monophosphorylation reac- arnesyl pyrophosphate (F-P-P) is a key intermediate in the tions. isoprenoid biosynthetic pathway positioned at a putative F Materials and Methods regulatory point capable of diverting isoprene units to a number 3 ͞ 3 of branch pathways (1). For example, squalene synthase activity, Materials. t,t-[ H]Farnesol (60 Ci mmol), t,t-[ H]farnesyl mono- ͞ 3 like HMG-CoA reductase, is down-regulated in mammalian cells phosphate (15 Ci mmol), t,t-[ H]farnesyl pyrophosphate (15 ͞ 3 ͞ ␥ 32 supplied with exogenous sterols (2–4). In Solanaceous plant cells Ci mmol), t,t,t-[ H]geranylgeraniol (60 Ci mmol), [ - P]GTP Ͼ ͞ ␥ 32 Ͼ ͞ responding to pathogen or elicitor challenge, sterol biosynthesis ( 3,000 Ci mmol), and [ - P]ATP ( 6,000 Ci mmol) were is arrested, and the cells synthesize and secrete antimicrobial obtained from American Radiolabeled Chemicals (St. Louis). 3 ͞ sesquiterpenes (5, 6). The reduction in sterol biosynthesis has t,t,t-[ H]Geranylgeranyl pyrophosphate (19.3 Ci mmol) was ob- ͞ been correlated with a suppression in squalene synthase activity, tained from DuPont NEN. Reverse-phase octadecyl (C18) silica whereas the induced accumulation of sesquiterpenes correlates gel plates were obtained from J. T. Baker. Benzyl-DEAE Ј with the activation of unique sesquiterpene biosynthetic enzyme cellulose, baker’s yeast nucleoside 5 diphosphate kinase, bac- activities (6, 7). Farnesylated and geranylgeranylated proteins terial alkaline phosphatase (Type III-S), and Merck precoated also have been documented extensively in both plant and animal cell types, and the requisite farnesyl- and geranylgeranyl- This paper was submitted directly (Track II) to the PNAS office. transferase activities have been described (8–11). Abbreviations: F-P-P, farnesyl pyrophosphate; F-OH, farnesol; SQ, squalestatin 1; GG-OH, Farnesol (F-OH) recently has received considerable attention geranylgeraniol; GG-P-P, geranylgeranyl pyrophosphate. as another important intracellular metabolite of isoprenoid ‡To whom reprint requests should be addressed. E-mail: [email protected] or metabolism. Early work on the feedback regulation of sterol [email protected]. biosynthesis suggested that both a sterol and a nonsterol me- The publication costs of this article were defrayed in part by page charge payment. This tabolite derived from mevalonate were necessary for the down- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. regulation of HMG-CoA reductase activity (2). Subsequent §1734 solely to indicate this fact. 13080–13085 ͉ PNAS ͉ November 9, 1999 ͉ vol. 96 ͉ no. 23 Downloaded by guest on September 28, 2021 silica gel G 60 thin-layer plates were obtained from Sigma. F-OH Kinase, F-P Kinase, and GG-OH Kinase Assays. Enzymatic assay Squalestatin was provided by Glaxo Wellcome. All other sol- mixtures for F-OH, F-P, and GG-OH kinase activities contained vents and chemicals were reagent grade and purchased from tobacco cell membranes (10–250 ␮g protein), 5 mM Tris⅐HCl ␮ standard commercial sources. (pH 7.4), 50–200 M CTP, 2–5 mM MgCl2, 0.05% CHAPS, 10 mM sodium orthovanadate, and 0.01–0.1 ␮Ci of either t,t-[3H]F- Synthesis of [␥-32P]CTP. [␥-32P]CTP was synthesized enzymatically OH (60 Ci͞mmol), t,t-[3H]F-P (15 Ci͞mmol), or t,t,t-[3H]GG- from CDP and [␥-32P]ATP by using baker’s yeast nucleoside 5Ј OH (60 Ci͞mmol) in a total volume of 20 ␮l. The kinase diphosphate kinase (N-3380; Sigma) as described previously (19). reactions were terminated after 10 min at 37° by the addition of ␮ Enzymatic reactions contained 80 mM CDP, 10 mM MgCl2,80mM 80 lofCH3OH. After centrifugation for 1 min in a microcen- Tris⅐HCl (pH 7.4), 4 units of yeast nucleoside 5Ј diphosphate kinase, trifuge, the supernatant was removed and the pellet was resus- ␮ ͞ and 1 mCi [␥-32P]ATP in a total volume of 0.05 ml. After 1 hr at pended in 50 lCH3OH H2O (4:1). After a second centrifuga- room temperature, the reaction mixture was chromatographed on tion, the supernatants were combined and dried under N2 gas. a column of Benzyl-DEAE cellulose (1.5 cm ϫ 15 cm) in 10 mM The labeled products were dissolved in a small volume of ␥ 32 NH4HCO3.[ - P]CTP was eluted from the benzyl-DEAE column water-saturated butanol and analyzed chromatographically by with a linear gradient (250 ml) of 0–0.5 M NH HCO . Fractions applying aliquots of each sample to Silica G 60 thin-layer plates 4 3 ͞ ͞ containing [␥-32P]CTP were combined, concentrated to a volume and developing with isopropanol NH4OH H2O (6:3:1). Radio- of 1 ml by rotary evaporation under reduced pressure at 30°C, and active products were detected by using a Bioscan Imaging System desalted by gel-filtration chromatography on a column (1.5 cm ϫ 30 (Washington, DC), and the level of kinase activity was calculated cm) of Sephadex G-10 eluted with distilled water. Fractions con- as a percentage of radiolabeled substrate (F-OH, F-P, or GG- taining [␥-32P]CTP were combined, concentrated again, and stored OH) converted to product (F-P, F-P-P, GG-P, GG-P-P). Enzy- 32 at Ϫ20°C. matic reactions containing [ P]NTPs as the isotopic substrate contained unlabeled isoprenyl alcohols (20 ␮M) and 0.1–0.5 ␮Ci ␥ 32 ␥ 32 ␥ 32 Cell Cultures and [3H]Farnesol-Labeling Studies.
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