Interplay between the cis-prenyltransferases and polyprenol reductase in the yeast Saccharomyces cerevisiae

Dolichol formation is examined in three Saccharomyces cerevisiae strains with mutations in the ERG20 gene encoding farnesyl diphosphate synthase (mevalonic acid pathway) and/or the ERG9 gene encoding squalene synthase (sterol synthesis pathway) differing in the amount and chain length of the polyisoprenoids synthesized. Our results suggest that the activities of two yeast cis-prenyltransferases Rer2p and Srt1p and polyprenol reductase are not co-regulated and that reductase may be the rate-limiting enzyme in dolichol synthesis if the amount of polyisoprenoids synthesized exceeds a certain level. We demonstrate that reductase preferentially acts on typical polyprenols with 13-18 isoprene residues but can reduce much longer polyprenols with even 32 isoprene residues.     

Precise bacterial polyprenol length control fails in Saccharomyces cerevisiae

A comparison of amino acid sequences of yeast Rer2p and Srt1p Z-prenyltransferases shows that the spatial organization of their substrate tunnels agrees with that determined by X-ray for the E. coli undecaprenyl diphosphate synthase (UPPs). The observed trend in the maxima of product length distribution shifted from C(55) in UPPs to C(80) in Rer2p and to C(110) in Srt1p. This suggests a significant increase in the size of the enzyme hydrophobic tunnel from approximately 1000 A(3) of E. coli UPPs to approximately 1300 A(3) required to accommodate C(80) in Rer2p and to 1700 A(3) for C(110) in Srt1p. Moreover, Srt1p products reaching C(290) indicate the failure of a strict bacterial-like chain length control. On the basis of E. coli UPPs crystallographic structure the yeast Rer2p model was constructed. In the model three amino acid residues inserted into the sequence corresponding to the "floor" region of the tunnel extends the bottom loop what results in the required increase of the tunnel volume. Moreover, thermal fluctuations of this loop occasionally create a hole in the tunnel floor, making escape of polyprenol omega end out of the tunnel possible what switches off the control mechanism of product length thereby allowing a practically unlimited elongation process leading to an exponential distribution of longer chain polyprenols.     

The Escherichia coli homologue of yeast RER2, a key enzyme of dolichol synthesis, is essential for carrier lipid formation in bacterial cell wall synthesis

We found in the Escherichia coli genome sequence a homologue of RER2, a Saccharomyces cerevisiae gene required for proper localization of an endoplasmic reticulum protein, and designated it rth (RER2 homologue). The disruption of this gene was lethal for E. coli. To reveal its biological function, we isolated temperature-sensitive mutants of the rth gene. The mutant cells became swollen and burst at the nonpermissive temperature, indicating that their cell wall integrity was defective. Further analysis showed that the mutant cells were deficient in the activity of cis-prenyltransferase, namely, undecaprenyl diphosphate synthase, a key enzyme of the carrier lipid formation of peptidoglycan synthesis. The cellular level of undecaprenyl phosphate was in fact markedly decreased in the mutants. These results are consistent with the fact that the Rer2 homologue of Micrococcus luteus shows undecaprenyl diphosphate synthase activity (N. Shimizu, T. Koyama, and K. Ogura, J. Biol. Chem. 273:19476-19481, 1998) and demonstrate that E. coli Rth is indeed responsible for the maintenance of cell wall rigidity. Our work on the yeast rer2 mutants shows that they are defective in the activity of cis-prenyltransferase, namely, dehydrodolichyl diphosphate synthase, a key enzyme of dolichol synthesis. Taking these data together, we conclude that the RER2 gene family encodes cis-prenyltransferase, which plays an essential role in cell wall biosynthesis in bacteria and in dolichol synthesis in eukaryotic cells and has been well conserved during evolution.     

The Yeast RER2 Gene, Identified by Endoplasmic Reticulum Protein Localization Mutations, Encodes cis-Prenyltransferase, a Key Enzyme in Dolichol Synthesis

As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfα1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. USA 90:8179–8183, 1993). The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general. The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes. RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal. RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution. Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis. This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself. The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol.     

The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N -glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae , we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis -prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2 , only HMG2 overexpression was able to restore growth of the yta7 Δ cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis.     

In vivo interaction between the human dehydrodolichyl diphosphate synthase and the Niemann-Pick C2 protein revealed by a yeast two-hybrid system

Dehydrodolichyl diphosphate (DedolPP) synthase catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize DedolPP, a biosynthetic precursor for dolichol which plays an important role as a sugar-carrier lipid in the biosynthesis of glycoprotein in eukaryotic cells. During certain pathological processes like Alzheimer's disease or some neurological disorders, dolichol has been shown to accumulate in human brain. In order to understand the regulatory mechanism of dolichol in eukaryotes, we performed a yeast two-hybrid screen using full length human DedolPP synthase gene [Endo et al. BBA 1625 (2003) 291] as a bait to find some proteins specifically interacting with the enzyme. We identified Niemann-Pick Type C2 protein (NPC2) to show a specific interaction with human DedolPP synthase. This interaction was further confirmed by in vitro co-immunoprecipitation experiment, indicating the possible physiological interaction between NPC2 and DedolPP synthase proteins in human.     

A novel family of longer chain length dolichols present in oleate-induced yeast Saccharomyces cerevisiae

The typical size of the yeast dolichol family ranges from 14 to 19 isoprene units D((14-19)) with dolichol(16) being the dominating species. Induction of peroxisome proliferation by growing the cells in medium containing oleate as carbon source induces the synthesis of an additional family of longer dolichols D((19-24)) with D(21) being the most prominent. This phenomenon is abolished in the peroxisome biogenesis deficient strain in which the PEX1 gene (encoding Pex1p peroxin) has been disrupted. The total amount of dolichols in pex1Delta cells is lower than in the wild-type cells, as is the amount of phosphatidylcholine. Moreover, the levels of 3-hydroxy-3-methylglutaryl CoA reductase and farnesyl diphosphate synthase, two key enzymes in dolichol biosynthesis, are decreased in the absence of a functional PEX1 gene. The presence of longer dolichols in oleate-induced Saccharomyces cerevisiae cells, the absence of this additional family in peroxisome deficient cells, and a decrease of the total amount of dolichols in these cells indicate the involvement of peroxisomes in the biosynthesis of dolichols in this organism.     

Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity

We report on the cDNA cloning and characterization of a novel short-chain isoprenyl diphosphate synthase from the aphid Myzus persicae. Of the three IPPS cDNAs we cloned, two yielded prenyltransferase activity following expression in Escherichia coli; these cDNAs encode identical proteins except for the presence, in one of them, of an N-terminal mitochondrial targeting peptide. Although the aphid enzyme was predicted to be a farnesyl diphosphate synthase by BLASTP analysis, rMpIPPS, when isopentenyl diphosphate and dimethylallyl diphosphate are supplied as substrates, typically generated geranyl diphosphate (C10) as its main product, along with significant quantities of farnesyl diphosphate (C15). Analysis of an MpIPPS homology model pointed to substitutions that could confer GPP/FPP synthase activity to the aphid enzyme.     

Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis

Farnesyl diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. In plants the presence of farnesyl diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized farnesyl diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl diphosphate over dimethylallyl diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl diphosphate and lavandulyl diphosphate, when incubated with dimethylallyl diphosphate and an additional product, the regular monoterpene geranyl diphosphate, when incubated with isopentenyl diphosphate and dimethylallyl diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.     

Cloning,expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.     

Identification of novel mammalian squalene synthase inhibitors using a three-dimensional pharmacophore

Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerisation of farnesyl diphosphate in a [1-4] head to head fashion to form squalene, and is the first committed step in cholesterol biosynthesis. Specific inhibitors of squalene synthase would inhibit cholesterol formation and allow production of other important compounds derived from the cholesterol biosynthetic pathway, namely the ubiquinones (co-enzyme Q(10)), dolichol, and would also allow the isoprenylation process of ras by farnesyl-protein transferase. The construction of a hypothetical squalene synthase three-dimensional pharmacophore is presented. It serves as a template for the identification of several new potential classes of inhibitors. The synthesis, anti-microbial and mammalian pig liver squalene synthase activities of analogues based on the bicyclo[3.2.0]heptane and bicyclo[3.3.0]octane ring systems are reported. Analogues of the latter system are pro-drug type inhibitors and exhibit promising biological activity.     

Studies on geranylgeranyl diphosphate synthase from rat liver: specific inhibition by 3-azageranylgeranyl diphosphate.

Geranylgeranyl diphosphate synthase from rat liver was separated from farnesyl diphosphate synthase, the most abundant and widely occurring prenyltransferase, by DEAE-Toyopearl column chromatography. The enzyme catalyzed the formation of E,E,E-geranylgeranyl diphosphate (V) from isopentenyl diphosphate (II) and dimethylallyl diphosphate (I), geranyl diphosphate (III), or farnesyl diphosphate (IV) with relative velocities of 0.09:0.15:1. 3-Azageranylgeranyl diphosphate (VII), designed as a transition-state analog for the geranylgeranyl diphosphate synthase reaction, was synthesized and found to act as a specific inhibitor for this synthase, but not for farnesyl diphosphate synthase. Diphosphate V and its Z,E,E-isomer (VI) also inhibited geranylgeranyl diphosphate synthase, but the effect was not as striking as that of the aza analog VII. Specific inhibition of geranylgeranyl diphosphate synthase by VII was also observed in experiments with 100,000g supernatants of rat brain and liver homogenates which contained isopentenyl diphosphate isomerase and prenyltransferases including farnesyl diphosphate synthase as well as geranylgeranyl diphosphate synthase. For farnesyl:protein transferase from rat brain, however, the aza compound did not show a stronger inhibitory effect than E,E,E-geranylgeranyl diphosphate.     

Overexpression of the Saccharomyces cerevisiae RER2 gene in Trichoderma reesei affects dolichol dependent enzymes and protein glycosylation

Protein secretion in Trichoderma reesei could be stimulated by overexpression of the yeast Saccharomyces cerevisiae DPM1 gene encoding dolichyl phosphate mannose synthase (DPMS) a key enzyme in the O-glycosylation pathway. The secreted proteins were glycosylated to the wild type level. On the other hand, the elevated concentration of GDP-mannose, a direct substrate for DPMS, resulting from overexpression in T. reesei of the mpg1 gene coding for guanyltransferase, did not affect secretion of proteins but did affect the degree of their O- and N-glycosylation. In this paper, we examined the effects of dolichol, an indispensable carrier of sugar residues in protein glycosylation, on the synthesis of glycosylated proteins. An increase in dolichol synthesis was obtained by overexpression of the yeast gene encoding cis-prenyltransferase, the first enzyme of the mevalonate pathway committed to dolichol biosynthesis. We observed that, an increased concentration of dolichol resulted in an increased expression of the dpm1 gene and DPMS activity and in overglycosylation of secreted proteins.     

DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate.

Biosynthesis of glycosylphosphatidylinositol and N-glycan precursor is dependent upon a mannosyl donor, dolichol phosphate-mannose (DPM). The Thy-1negative class E mutant of mouse lymphoma and Lec15 mutant Chinese hamster ovary (CHO) cells are incapable of DPM synthesis. The class E mutant is defective in the DPM1 gene which encodes a mammalian homologue of Saccharomyces cerevisiae Dpm1p that is a DPM synthase, whereas Lec15 is a different mutant, indicating that mammalian DPM1 is not sufficient for DPM synthesis. Here we report expression cloning of a new gene, DPM2, which is defective in Lec15 cells. DPM2, an 84 amino acid membrane protein expressed in the endoplasmic reticulum (ER), makes a complex with DPM1 that is essential for the ER localization and stable expression of DPM1. Moreover, DPM2 enhances binding of dolichol phosphate, a substrate of DPM synthase. Mammalian DPM1 is catalytic because a fusion protein of DPM1 that was stably expressed in the ER synthesized DPM without DPM2. Therefore, biosynthesis of DPM in mammalian cells is regulated by DPM2.     

An alternative cis-isoprenyltransferase activity in yeast that produces polyisoprenols with chain lengths similar to mammalian dolichols

Dolichyl monophosphate (Dol-P) is a polyisoprenoid glycosyl carrier lipid essential for the assembly of a variety of glycoconjugates in the endoplasmic reticulum of eukaryotic cells. In yeast, dolichols with chain lengths of 14--17 isoprene units are predominant, whereas in mammalian cells they contain 19--22 isoprene units. In this biosynthetic pathway, t,t-farnesyl pyrophosphate is elongated to the appropriate long chain polyprenyl pyrophosphate by the sequential addition of cis-isoprene units donated by isopentenyl pyrophosphate with t,t,c-geranylgeranyl pyrophosphate being the initial intermediate formed. The condensation steps are catalyzed by cis-isoprenyltransferase (cis-IPTase). Genes encoding cis-IPTase activity have been identified in Micrococcus luteus, Escherichia coli, Arabidopsis thaliana, and Saccharomyces cerevisiae (RER2). Yeast cells deleted for the RER2 locus display a severe growth defect, but are still viable, possibly due to the activity of an homologous locus, SRT1. The dolichol and Dol-P content of exponentially growing revertants of RER2 deleted cells (Delta rer2) and of cells overexpressing SRT1 have been determined by HPLC analysis. Dolichols and Dol-Ps with 19--22 isoprene units, unusually long for yeast, were found, and shown to be utilized for the biosynthesis of lipid intermediates involved in protein N-glycosylation. In addition, cis-IPTase activity in microsomes from Delta rer2 cells overexpressing SRT1 was 7- to 17-fold higher than in microsomes from Delta rer2 cells. These results establish that yeast contains at least two cis-IPTases, and indicate that the chain length of dolichols is determined primarily by the enzyme catalyzing the chain elongation stage of the biosynthetic process.     

Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyses have suggested that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early region of the Golgi apparatus. The rer1 mutant we isolated in a previous study mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene and determined its reading frame. RER1 encodes a hydrophobic protein of 188 amino acid residues containing four putative membrane spanning domains. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER, implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an epitope derived from the influenza hemagglutinin was added to the C-terminus of Rer1p and the cells expressing this tagged but functional protein were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern that is typical for Golgi proteins and clearly distinct from the ER staining. This punctate staining was in fact exaggerated in the sec7 mutant that accumulates the Golgi membranes at the restrictive temperature. Furthermore, double staining of Rer1p and Ypt1p, a GTPase that is known to reside in the Golgi apparatus, showed good colocalization. Subcellular fractionation experiments indicated that the fractionation pattern of Rer1p was similar to that of an early Golgi protein, Och1p. From these results, we suggest that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.     

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.     

Cell growth-dependent coordination of lipid signaling and glycosylation is mediated by interactions between Sac1p and Dpm1p

The integral membrane lipid phosphatase Sac1p regulates local pools of phosphatidylinositol-4-phosphate (PtdIns(4)P) at endoplasmic reticulum (ER) and Golgi membranes. PtdIns(4)P is important for Golgi trafficking, yet the significance of PtdIns(4)P for ER function is unknown. It also remains unknown how localization of Sac1p to distinct organellar membranes is mediated. Here, we show that a COOH-terminal region in yeast Sac1p is crucial for ER targeting by directly interacting with dolicholphosphate mannose synthase Dpm1p. The interaction with Dpm1p persists during exponential cell division but is rapidly abolished when cell growth slows because of nutrient limitation, causing translocation of Sac1p to Golgi membranes. Cell growth-dependent shuttling of Sac1p between the ER and the Golgi is important for reciprocal control of PtdIns(4)P levels at these organelles. The fraction of Sac1p resident at the ER is also required for efficient dolichol oligosaccharide biosynthesis. Thus, the lipid phosphatase Sac1p may be a key regulator, coordinating the secretory capacity of ER and Golgi membranes in response to growth conditions.     

Farnesyl diphosphate synthase is a cytosolic enzyme in Leishmania major promastigotes and its overexpression confers resistance to risedronate

Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.