PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots

Tanshinones, a group of active ingredients in Salvia miltiorrhiza, are derived from at least two biosynthetic pathways, which are the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the plastids. Abscisic acid (ABA) and methyl jasmonate (MJ) are two well-known plant hormones induced by water stress. In this study, effects of polyethylene glycol (PEG), ABA and MJ on tanshinone production in S. miltiorrhiza hairy roots were investigated, and the role of MJ in PEG- and ABA-induced tanshinone production was further elucidated. The results showed that tanshinone production was significantly enhanced by treatments with PEG, ABA and MJ. The mRNA levels of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS), as well as the enzyme activities of HMGR and DXS were stimulated by all three treatments. PEG and ABA triggered MJ accumulation. Effects of PEG and ABA on tanshinone production were completely abolished by the ABA biosynthesis inhibitor [tungstate (TUN)] and the MJ biosynthesis inhibitor [ibuprofen (IBU)], while effects of MJ were almost unaffected by TUN. In addition, MJ-induced tanshinone production was completely abolished by the MEP pathway inhibitor [fosmidomycin (FOS)], but was just partially arrested by the MVA pathway inhibitor [mevinolin (MEV)]. In conclusion, a signal transduction model was proposed that exogenous applications of PEG and ABA triggered endogenous MJ accumulation by activating ABA signaling pathway to stimulate tanshinone production, while exogenous MJ could directly induce tanshinone production mainly via the MEP pathway in S. miltiorrhiza hairy roots.     

Influence of mevinolin on chloroplast terpenoids in Cannabis sativa

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In this study, we investigated the inhibitory effect of the MVA pathway on isoprenoid biosynthesized by the MEP pathway in Cannabis sativa by treatment with mevinolin. The amount of chlorophyll a, b, and total showed to be significantly enhanced in treated plants in comparison with control plants. Also, mevinolin induced the accumulation of carotenoids and α-tocopherol in treated plants. Mevinolin caused a significant decrease in tetrahydrocannabinol (THC) content. This result show that the inhibition of the MVA pathway stimulates MEP pathway but none for all metabolites.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.     

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

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 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.     

A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway

Plants are able to integrate exogenous 1-deoxy-D-xylulose (DX) into the 2C-methyl-D-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-D-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing D-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as "xylulose kinase" might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta.     

Effects of Gibberellic Acid on Primary Terpenoids and △9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration,and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP)pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA3) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA3 caused a decrease in DXS activity in both sexes that it wasaccompanied by a decrease in chlorophylls, carotenoids and △9-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA3 showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants.The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA3 can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

HMG-CoA reductase limits artemisinin biosynthesis and accumulation in <Emphasis Type="Italic">Artemisia annua</Emphasis> L. plants

In vivo modulation of HMG-CoA reductase (HMGR) activity and its impact on artemisinin biosynthesis as well as accumulation were studied through exogenous supply of labeled HMG-CoA (substrate), labeled MVA (the product), and mevinolin (the competitive inhibitor) using twigs of Artemisia annua L. plants collected at the pre-flowering stage. By increasing the concentration (2–16 μM) of HMG-CoA (3-¹⁴C), incorporation of labeled carbon into artemisinin was enhanced from 7.5 to 17.3 nmol (up to 130%). The incorporation of label (¹⁴C) into MVA and artemisinin was inhibited up to 87.5 and 82.9%, respectively, in the presence of 200 μM mevinolin in incubation medium containing 12 μM HMG-CoA (3-¹⁴C). Interestingly, by increasing the concentration of MVA (2-¹⁴C) from 2 to 18 μM, incorporation of label (¹⁴C) into artemisinin was enhanced from 10.5 to 35 nmol (up to 233%). When HMG-CoA (3-¹⁴C) concentration was increased from 12 to 28 μM in the presence of 150 μM mevinolin, the inhibitions in the incorporation of label (¹⁴C) into MVA and artemisinin were, however, reversed and the labels were found to approach their values in twigs fed with 12 μM HMG-CoA (3-¹⁴C) without mevinolin. In another experiment, 14.2% inhibition in artemisinin accumulation was observed in twigs in the presence of 175 μM fosmidomycin, the competitive inhibitor of 1-deoxy-d-xylulose 5-phosphate reductase (DXR). HMG-CoA reductase activity and artemisinin accumulation were also increased by 18.6 to 24.5% and 30.7 to 38.4%, respectively, after 12 h of treatment, when growth hormones IAA (100 ppm), GA₃ (100 ppm) and IAA + GA₃ (50 + 50 ppm) were sprayed on A. annua plants at the pre-flowering stage. The results obtained in this study, hence, demonstrate that the mevalonate pathway is the major contributor of carbon supply to artemisinin biosynthesis and HMGR limits artemisinin synthesis and its accumulation in A. annua plants.     

Differential interaction of branch-specific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), which catalyzes the formation of mevalonic acid (MVA), can be specifically blocked by mevinolin. Inhibition of HMGR in vivo leads to an arrest in cell cycle progression in tobacco ( Nicotiana tabacum L. Bright Yellow 2) cells. As MVA in plants is the common precursor of a myriad of isoprenoid products synthesized in the cytosol and mitochondria, it is difficult to identify among such MVA-dependent molecules those whose lack may lead to cell cycle arrest. In an attempt to do so, branch-specific inhibitors of the cytosolic isoprenoid pathway downstream from MVA were used to study their capacity to block cell cycle progression. The effects of squalestatin (sterol biosynthesis inhibitor), chaetomellic acid A and patulin (protein prenyltransferase (PT) inhibitors) and tunicamycin (inhibitor of dolichol-dependent protein glycosyl transferase, thus mimicking the effect of an absence of dolichol) were compared to those induced by mevinolin. In this way, squalestatin and chaetomellic acid were identified as behaving like true cell cycle inhibitors, in that they led to a specific arrest in the cell cycle. However, they did not exactly mimic the mevinolin-induced effects. Patulin proved to be of high general toxicity, which suggests that it may affect other reactions besides blockage of protein isoprenylation. Finally, tunicamycin efficiently blocked growth of cell suspension cultures, but did not arrest the cells in a specific phase of the cell cycle. Results are discussed in the context of a better understanding of the essential implication of isoprenoids in plant cell cycle progression.     

Farnesol-induced cell death and stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in tobacco cv bright yellow-2 cells

Growth inhibition of tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells by mevinolin, a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) could be partially overcome by the addition of farnesol. However, farnesol alone inhibited cell division and growth as measured by determination of fresh weight increase. When 7-d-old tobacco cv Bright Yellow-2 cells were diluted 40-fold into fresh culture, the cells exhibited a dose-dependent sensitivity to farnesol, with 25 microM sufficient to cause 100% cell death, as measured by different staining techniques, cytometry, and monitoring of fragmentation of genomic DNA. Cells were less sensitive to the effects of farnesol when diluted only 4-fold. Farnesol was absorbed by the cells, as examined by [1-(3)H]farnesol uptake, with a greater relative enrichment by the more diluted cells. Both mevinolin and farnesol treatments stimulated apparent HMGR activity. The stimulation by farnesol was also reflected in corresponding changes in the steady-state levels of HMGR mRNA and enzyme protein with respect to HMGR gene expression and enzyme protein accumulation.     

Biosynthesis of the iridoid glucoside,lamalbid, in Lamium barbatum

The biosynthesis of the iridoid glucoside lamalbid in Lamium barbatum, a plant species in the Lamiaceae, was investigated by administrating ¹³C-labeled intermediates of MVA and MEP pathways, respectively. The results demonstrated that [3,4,5-¹³C₃]1-deoxy-d-xylulose 5-phosphate could be incorporated into lamalbid, whereas the incorporation of [2-¹³C₁]mevalonolactone was not observed. Based on the ¹³C labeling pattern of lamalbid and the incorporation data, we deduce that the iridoid glucoside in L. barbatum is biosynthesized through the MEP pathway, whereas the classic MVA pathway is not utilized.     

Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In at least some plants (including Arabidopsis), common precursors are exchanged between the cytosol and the plastid. However, little is known about the signals that coordinate their biosynthesis and exchange. To identify such signals, we arrested seedling development by specifically blocking the MVA pathway with mevinolin (MEV) or the MEP pathway with fosmidomycin (FSM) and searched for MEV-resistant Arabidopsis mutants that also could survive in the presence of FSM. Here, we show that one such mutant, rim1, is a new phyB allele (phyB-m1). Although the MEV-resistant phenotype of mutant seedlings is caused by the upregulation of MVA synthesis, its resistance to FSM most likely is the result of an enhanced intake of MVA-derived isoprenoid precursors by the plastid. The analysis of other light-hyposensitive mutants showed that distinct light perception and signal transduction pathways regulate these two differential mechanisms for resistance, providing evidence for a coordinated regulation of the activity of the MVA pathway and the crosstalk between cell compartments for isoprenoid biosynthesis during the first stages of seedling development.     

Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells

Mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, was used to study the importance of mevalonic acid (MVA) for cell cycle progression of tobacco (Nicotiana tabacum L.) BY-2 cells. After treatment with 5 microM mevinolin, the cell cycle progression was completely blocked and two cell populations accumulated (80% in phase G0/G1 and 20% in G2/M). The arrest could be released by subsequent addition of MVA. Effects were compared to those caused by aphidicolin, an inhibitor of alpha-like DNA polymerases that blocks cell cycle at the entry of the S phase. The 80% proportion of mevinolin-treated TBY-2 cells was clearly arrested before the aphidicolin-inducible block. By the aid of a double-blocking technique, it was shown that the mevinolin-induced cell arrest of highly synchronized cells was due to interaction with a control point located at the mitotic telophase/entry G1 phase. Depending on the developmental stage, mevinolin induced rapid cell death in a considerable percentage of cells. Mevinolin treatment led to a partial synchronization, as shown by the increase in mitotic index. The following decrease was correlated with the above-mentioned induction of cell death.     

Isoprenoid biosynthesis via a mevalonate-independent pathway in plants: cloning and heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint

Two distinct pathways are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids. The classical acetate/mevalonate pathway operates in the cytosol, whereas plastidial isoprenoids originate via a novel mevalonate-independent route that involves a transketolase-catalyzed condensation of pyruvate and D-glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate as the first intermediate. Based on in vivo feeding experiments, rearrangement and reduction of deoxyxylulose phosphate have been proposed to give rise to 2-C-methyl-D-erythritol-4-phosphate as the second intermediate of this pyruvate/glyceraldehyde-3-phosphate pathway (1-3). The cloning of an Escherichia coli gene encoding an enzyme capable of converting 1-deoxy-D-xylulose-5-phosphate to 2-C-erythritol-4-phosphate was recently reported (4). A cloning strategy was developed for isolating the gene encoding a plant homolog of this enzyme from peppermint (Mentha x piperita), and the identity of the resulting cDNA was confirmed by heterologous expression in E. coli. Unlike the microbial reductoisomerase, the plant ortholog encodes a preprotein bearing an N-terminal plastidial transit peptide that directs the enzyme to plastids where the mevalonate-independent pathway operates in plants. The peppermint gene comprises an open reading frame of 1425 nucleotides which, when the plastidial targeting sequence is excluded, encodes a deduced enzyme of approximately 400 amino acid residues with a mature size of about 43.5 kDa.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis

Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting.     

The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.