Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions

Abstract

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [(3)H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [(3)H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [(3)H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [(3)H]F-OH or [(3)H]geranylgeraniol ([(3)H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [(3)H]F-P and [(3)H]F-P-P were synthesized when exogenous [(3)H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor-product relationship between [(3)H]F-P and [(3)H]F-P-P. In agreement with this kinetic pattern of labeling, [(32)P]F-P and [(32)P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [gamma-(32)P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [(3)H]GG-OH to [(3)H]geranylgeranyl monophosphate and [(3)H]geranylgeranyl pyrophosphate ([(3)H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [(3)H]CTP was formed when microsomes were incubated with [(3)H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation.

Figure 1

SQ inhibits the incorporation of [³H]F-OH into sterols by N. tabacum cell cultures (A) and microsomal squalene synthase activity measured in vitro (B). Cell cultures were incubated with the indicated concentrations of SQ for 6.5 hr before adding 0.5 μCi [³H]F-OH for an additional 4.5 hr. The incorporation of radiolabel into digitonin-precipitable sterols by control cells (3,000 dpm) is compared with that for the SQ-treated cells (A). The effect of SQ on the in vitro squalene synthase activity (100% = 40 nmol/hr⋅mg protein) was determined after preincubating microsomes for 15 min with the indicated concentrations of SQ (B).

Figure 2

[³H]F-OH and [³H]GG-OH are incorporated into N. tabacum cell proteins. Cell cultures were pretreated with 5 μM lovastatin for 16 hr before incubating with either [³H]F-OH (A) or [³H]GG-OH (B) for 8 hr. Total, radiolabeled proteins were extracted in SDS sample buffer, size-separated by SDS/PAGE, and visualized by fluorography.

Figure 3

Time course for the CTP-mediated phosphorylation of [³H]F-OH catalyzed by membrane fractions from N. tabacum cells. The reaction mixtures and the procedure for assaying the formation of [³H]F-P and [³H]F-P-P are described in Materials and Methods.

Figure 4

Enzymatic transfer of [³²P]phosphoryl groups from [γ-³²P]CTP to exogenous F-OH (B) and F-P (C). Reaction mixtures contained 5 mM Tris⋅HCl, pH 7.4, tobacco microsomes (30 μg protein), 0.05% CHAPS, 5 mM MgCl₂, 1 mM EDTA, 10 mM sodium orthovanadate, 1 μM [γ-³²P]CTP (20,000 cpm/pmol), and either no exogenous substrate (A), 2 μM F-OH (B), or 2 μM F-P (C) in a total volume of 20 μl. After incubation at 37°C for 10 min, the reaction was stopped by the addition of 0.5 ml of H₂O-saturated n-butanol. The butanol extract was washed three times with 0.5 ml of n-butanol-saturated H₂O to remove unreacted [γ-³²P]CTP and dried under a stream of N₂. The enzymatically labeled products were identified by chromatography on Silica gel G plates developed with isopropanol/NH₄OH/H₂O (6:3:1). The chromatographic positions of authentic F-P and F-P-P are indicated by arrows.