sampangine inhibits heme biosynthesis in both yeast and ...heme synthesis likely by hyperactivating...

EUKARYOTIC CELL, Nov. 2011, p. 1536–1544 Vol. 10, No. 111535-9778/11/$12.00 doi:10.1128/EC.05170-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Sampangine Inhibits Heme Biosynthesis in both Yeast and Human�†Zhiwei Huang,1,6 Kaifu Chen,1,3 Tao Xu,4‡ Jianhuai Zhang,1 Yongxiang Li,1 Wei Li,3

Ameeta K. Agarwal,4 Alice M. Clark,4 John D. Phillips,5* and Xuewen Pan1,2*Verna and Marrs McLean Department of Biochemistry and Molecular Biology,1 Department of Molecular and Human Genetics,2

and Division of Biostatistics, Dan L. Duncan Cancer Center,3 Baylor College of Medicine, Houston, Texas 77030;National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University,

Mississippi 386774; Division of Hematology, Department of Medicine, University of Utah School ofMedicine, Salt Lake City, Utah 841325; and Institute of Biological Sciences and Biotechnology,

Donghua University, Shanghai 201620, People’s Republic of China6

Received 6 July 2011/Accepted 30 August 2011

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms.Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactiveoxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanismof action by sampangine and that increases in the levels of reactive oxygen species are secondary to hemedeficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomycescerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediateproducts of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through anunusual mechanism—by hyperactivating uroporhyrinogen III synthase—to inhibit heme biosynthesis. We alsoshow that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study alsoreveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electrontransport chain.

The plant-derived alkaloid sampangine has broad and po-tent antiproliferation activities against fungal pathogens, hu-man cancer cell lines, malaria parasites, and mycobacteria (19,20, 25, 31). In particular, its potency against various humanfungal pathogens in vitro is comparable to the potencies ofexisting antifungal drugs (1). Other analogs of the same apor-phine family of alkaloids have also been shown to exhibitantiproliferation activities against viruses, bacteria, fungi, par-asites, and tumor cell lines (7, 8, 17, 24, 34, 36). However, themechanism of action exhibited by sampangine and its analogsin both cancer cells and human pathogens remains unclear.Sampangine was shown to induce apoptosis in cancer cells bystimulating the generation of reactive oxygen species (ROS)(20). A closely related alkaloid, ascididemin, was shown toinhibit the growth of Mycobacterium tuberculosis through irondepletion (5). We have previously also shown that sampangineinhibits fungal growth, likely by interfering with heme metab-olism (1). This was supported by the observations that multiplemutants with mutations affecting the Saccharomyces cerevisiaeyeast heme biosynthesis pathway were comparatively more

sensitive to sampangine than a wild-type strain and that exog-enously supplied hemin partly suppressed the inhibitory activ-ity of the drug (1). However, exogenous hemin failed tocompletely reverse the inhibitory effect of sampangine. Over-expressing genes in the heme biosynthesis pathway also failedto confer sampangine resistance in a wild-type strain back-ground (unpublished results). These results raise the questionof whether heme synthesis is a primary target of the drug andalso whether there is any molecular relationship between themany different cellular effects of sampangine.

In this study, we took an unbiased functional genomic ap-proach by systematically screening the yeast genome-wide de-letion mutant libraries to identify mutants exhibiting hypersen-sitivity or resistance toward the drug. Although none of themutants tested conferred obvious resistance, we identified 132mutants that were hypersensitive. Among these, the most sen-sitive ones affected mitochondrial functions, especially sub-units of the ATP synthase. We next performed genome-widesynthetic lethality analyses with strains with two representativemutations (atp1� and yme1�) that conferred the highest drugsensitivity to identify common genes or pathways that, whendisrupted, cause severe growth defects or lethality. This iden-tified mutations that affect the electron transport chain (ETC)and heme synthesis (hem14�) to be among the most significantones. Comparison of synthetic lethality profiles suggested thatthe hem14� mutation reproduced the effects of sampanginetreatment much better than mutations in ETC (cox17� orcyc3�), suggesting that the heme biosynthesis pathway is aprimary target of the drug. Consistent with this model, wefound that sampangine inhibits heme production in both yeastand human cells. Moreover, our studies on porphyrin profilesand enzyme activity levels indicate that sampangine inhibits

* Corresponding author. Mailing address for John D. Phillips: Di-vision of Hematology, Department of Medicine, University of UtahSchool of Medicine, Salt Lake City, UT 84132. Phone: (801) 581-6650.Fax: (801) 585-3432. E-mail: [email protected]. Mailingaddress for Xuewen Pan: Department of Biochemistry and MolecularBiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX77030. Phone: (713) 798-3698. Fax: (713) 796-9438. E-mail: [email protected].

† Supplemental material for this article may be found at http://ec.asm.org/.

‡ Present address: Life Sciences Institute, 210 Washtenaw Avenue,University of Michigan, Ann Arbor, MI 48109.

� Published ahead of print on 9 September 2011.

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heme synthesis likely by hyperactivating Hem4, the fourth stepwithin the pathway. We also found that the growth defect of ahem14� yeast mutant was partially suppressed by the antioxi-dant N-acetyl cysteine (NAC), indicating that heme deficiencylikely accounts for the increased ROS levels observed in sam-pangine-treated cells (1, 20).

MATERIALS AND METHODS

Yeast media and chemicals. The haploid selection synthetic complete (SC)medium without Leu, His, and Arg and with G418 and L-canavanine (Can)contained dextrose (20 g/liter), yeast nitrogen base without amino acids andammonium sulfate (1.7 g/liter), SC medium Leu-His-Arg dropout mix (2 g/liter),sodium glutamate (1 g/liter), G418 (200 mg/liter), L-canavanine (60 mg/liter), andagar (2%). Sodium glutamate substituted for ammonium sulfate as the nitrogensource to make the G418 selection more reliable on the minimal medium. Aversion of this that lacked uracil was used to select for double mutants duringsynthetic lethality analyses. SC medium contained dextrose (20 g/liter), yeastnitrogen base without amino acids and ammonium sulfate (1.7 g/liter), SC me-dium mix (2 g/liter), and sodium glutamate (1 g/liter) with or without agar (2%).A similar medium that lacked uracil was also used. Solid sporulation mediumcontained potassium acetate (10 g/liter), zinc acetate (0.05 g/liter), and agar(2%).

ATP, N-acetyl cysteine, and antimycin were purchased from Sigma-Aldrichand dissolved in water as stock solutions of 1 M, 100 mg/ml, and 100 mg/ml,respectively. Sampangine was isolated as described previously (31) and dissolvedin dimethyl sulfoxide (DMSO) as a stock solution of 2 mg/ml.

Yeast strains and plasmids. Yeast strains used in this study were haploid-convertible heterozygous diploid yeast deletion mutants (mutants YSC4035 andYSC4428; Open Biosystems) and their haploid MATa convertants and were usedafter sporulation and haploid selection as previously described (29). The geno-type of a typical haploid strain was MATa ura3�0 leu2�0 his3�0 met15�0can1�::LEU2-MFA1pr-HIS3 goi�::kanMX, where goi� stands for deletion of anygene of interest. A ho�::kanMX mutant was used as a surrogate wild-type controlin most experiments because the HO gene is already mutated in the parent strain.

Wild-type genes HEM3, HEM4, and HEM12 were PCR amplified and clonedinto the vector YEplac195 (2�m, URA3) (13) to construct the overexpressionplasmids.

Screening for and validation of sampangine-hypersensitive haploid YKOs.Screening for and validation of sampangine-hypersensitive haploid yeast knock-outs (YKOs) were carried out essentially as previously described (28, 29). For thescreen, a pool of haploid-convertible heterozygote diploid yeast deletion mutantswas sporulated. Pools of isogenic MATa haploid cells were derived by growth ona haploid selection medium (SC medium without Leu, His, and Arg and withG418 and canavanine) that either contained (experiment) or lacked (control)sampangine at 0.5 �g/ml. Relative representation of each YKO in drug-treatedand untreated pools was compared by bar code microarray analysis. For valida-tion, individual haploid convertible heterozygous diploid mutants were sporu-lated, spotted onto haploid selection medium that either contained or lackedsampangine at the indicated concentrations, and incubated at 30°C for 3 days.The confirmed results are reported in Table S1 in the supplemental material.

Genome-wide synthetic lethality analyses and validation. Genome-wide syn-thetic lethality analyses and validation were carried out essentially as previouslydescribed (28, 29). The query constructs used in this study were atp1�::URA3,yme1�::URA3, hem14�::URA3, cox17�::URA3, and cyc3�::URA3, each of whichcontained �1.5-kb flanking sequences that allow highly efficient and accuratedisruption of the target gene. Genome-wide synthetic lethality screens wereperformed with the atp1�::URA3 and yme1�::URA3 constructs. The other con-structs were tested individually against the list of 132 mutations that conferredsampangine hypersensitivity.

Cellular component enrichment analysis. Gene lists identified from the ge-nome-wide sampangine hypersensitivity mutant profiling and synthetic lethalityanalyses were subject to Database for Annotation, Visualization, and IntegratedDiscovery (DAVID) analysis as previously described (16). Significantly enrichedcellular components were selected on the basis of a cutoff false discovery rate (Q)value of 0.05, and the fold enrichment was plotted.

Measuring heme levels in yeast. An overnight culture of a yeast strain carryingan empty plasmid or overexpressing one of the heme biosynthesis genes wasseeded into 50 ml of fresh SC medium without Ura at a 0.1 optical density at 600nm (OD600)/ml and incubated at 30°C for about 2 doublings. The culture wassplit into two, with one exposed to sampangine (1 �g/ml) and the other oneexposed to DMSO. Both cultures were subsequently incubated at 30°C for 3 h.

We note that sampangine at this concentration did not obviously reduce growthof the wild-type strain during this treatment period. Similar numbers of cellsfrom each culture were harvested, washed with ice-cold water, and homogenizedin 500 �l of lysis buffer as previously described (27). Two microliters of each celllysate was used to measure the amount of heme with a hemin assay kit (Bio-Vision, CA) according to the manufacturer’s instructions. The protein concen-tration of each lysate was also measured and used to calculate the heme con-centration as fmol/�g protein. Three independent experiments were performedfor each culture condition, and the results were averaged. The average hemelevel in the wild-type strain containing the empty vector that was grown in theabsence of sampangine was set at 100%.

Measuring heme levels in human cancer cell lines. Exponentially growingcultures (2 � 105) of the acute T cell leukemia Jurkat and non-small cell lungcancer NCI-H1299 cell lines were seeded into 6-well plates containing RPMI1640 supplemented with fetal bovine serum (10%; Sigma-Aldrich), glutamine (2mM), penicillin (100 U/ml), and streptomycin (100 �g/ml) and incubated at 37°Cin humidified air with 5% CO2. After an overnight incubation, cells were treatedwith sampangine (at 0.01 �g/ml or 0.1 �g/ml) or DMSO as the vehicle controland incubated for another 24 h, harvested, and washed softly with germfreephosphate-buffered saline. About 5,000 cells from each culture were used tomeasure the amount of hemin present using a hemin assay kit (BioVision, CA)according to the manufacturer’s instruction. At least three independent experi-ments were performed for each culture condition, and the results were averaged.The average heme level for each cell line grown in the absence of sampanginewas set at 100%.

Determination of porphyrin profile in yeast. An overnight culture of wild-typeyeast grown in liquid SC medium was used to inoculate 100 ml fresh medium at0.1 OD600/ml. After one doubling, either sampangine at the 50% inhibitoryconcentration (IC50; 1.17 �g/ml) or DMSO (0.25%) was added to the cultures.The cells were allowed to grow for 14 h after treatment, and equal numbers ofcells from the corresponding treated and untreated cultures were harvested bycentrifugation. The cells were washed once with sterile distilled water, and thecell pellets were flash frozen in liquid nitrogen. The experimental conditions,including the medium, temperature, aeration, and concentration of sampangine,were identical to the conditions used in a previously reported study (1). Threeindependent experiments were performed on independently grown cultures. Thesamples were processed for high-performance liquid chromatography (HPLC)analysis at Frontier Scientific Inc. (Logan, UT) as described previously (4).

In vitro assays of porphobilinogen deaminase (PBGD), uroporphyrinogen IIIsynthase (UIIIS), and uroporphyrinogen III decarboxylase (UroD). (i) PBGDassay. A 100-�l mixture contained 330 �M porphobilinogen (PBG), 70 mM Tris(pH 7.65), 10 mM dithiothreitol (DTT), 6 �g purified recombinant humanporphobilinogen deaminase (rPBGD), and 6 �l of stock DMSO that containedvarious amounts of sampangine. The mixture was then incubated at 37°C for 30min in the dark. The reaction was terminated by adding 100 �l of 3 M HCl. Theuroporphyrinogen I produced in the assay mixture was then oxidized to uropor-phyrin I by exposure to UV light for 30 min. The acidified sample was thencentrifuged in a microcentrifuge. The supernatant was analyzed by ultra-performance liquid chromatography (UPLC) as described below.

(ii) UIIIS assay. The UIIIS assay is performed as described above with theaddition of 2 �g of purified recombinant UIIIS (rUIIIS). The porphyrin isomerswere resolved by a method derived from those described previously (35, 39). AnHPLC system that consisted of a Waters 2795 separations module (Alliance HT),a Waters 474 scanning fluorescence detector, a Waters 2996 photodiode arraydetector and a Phenomenex Gemini C18 column (250 mm by 4.6 mm by 5 �m)was used. The solvents were 1 M NH4OH in water adjusted to pH 5.16 withglacial acetic acid (solvent A), methanol (solvent B), acetonitrile (solvent C), and10% (vol/vol) methanol in water (solvent D). All gradients were linear, and theflow rate was always 1 ml/min for the duration of the 40-min run. Solvent C wasset at 10% throughout the run. The gradient conditions were set at 75% solventA and 15% solvent B at 0 min, 60% solvent A and 30% solvent B at 8 min, 40%solvent A and 50% solvent B at 13 min, 28% solvent A and 62% solvent B at 17min, and 70% solvent B and 20% solvent D at 20 min and 25 min and then wentback to initial conditions at 28 min.

(iii) UroD assay. The UroD assay was carried out essentially as previouslydescribed (32). Uroporphyrinogen I (30 to 35 nmol) for a single assay wasenzymatically produced in a 100-�l mixture from PBG using 6 �g PBGD and 2�g rUIIIS as described above.

(iv) Separation and detection of porphyrins. About 10 �l of the supernatantfrom the assay described above was injected into a reverse-phase Waters AcquityUPLC system with an Acquity fluorescence detector that was set at 404 nmexcitation and 618 nm emission. A BEH phenyl 1.7-�m column (2.1 by 50 mm)was used to resolve the various porphyrins for a 3.5-min total run time at 60°C.

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The flow rate was set at 0.7 ml per min. Two solvents were used: solvent Aconsisted of 0.1% formic acid in water, while solvent B was pure acetonitrile. Thegradient conditions were set at 60% solvent A at 0.0 min, 35% solvent A at 2.0min, 10% solvent A at 2.1 min, and 1% solvent A at 2.6 min and then went backto initial conditions at 2.7 min. Except at 2.0 min, where the gradient was set atconvex 5, all the rest were set at linear 6. The millivolt signals detected for thevarious porphyrins were individually compared with those in a standard solutionthat contained 5 pmol each of 8-, 7-, 6-, 5-, 4-, and 2-carboxylporphyrin per 10-�linjection. The chromatograms were processed using Waters Empower Pro soft-ware.

RESULTS

Mutants of mitochondrial ATP synthase are hypersensitiveto sampangine. To identify the primary target of sampanginein a eukaryote, we first systematically screened the yeast ge-nome-wide heterozygous diploid deletion mutants (11) andfound that the mutant with the TOM40/tom40� mutation,which affects mitochondrial protein import (37), was the onlyone exhibiting significant hypersensitivity. However, this hyper-sensitivity was likely due to the intrinsic growth defects exhib-ited by this mutant (data not shown). We also screened agenome-wide open reading frame overexpression library thatwe constructed (Z. Huang and X. Pan, unpublished data) andfound that overexpressing the multidrug ABC transporterSNQ2 (33) confers sampangine resistance (data not shown),possibly due to decreased drug accumulation inside yeast cells.We next screened the genome-wide haploid deletion mutantswith complete loss of gene functions for increased drug sensi-tivity, reasoning that such mutants could define cellular func-tions closely related to the drug’s target, if not the target itself.

Upon individual validation, we identified 132 haploid deletionmutants that were significantly more sensitive to sampangine at0.5 �g/ml than a wild-type strain (see Table S1 in the supple-mental material). Highly enriched among these were mutantswith mutations affecting the mitochondrial ATP synthase, his-tone modification and chromatin remodeling, peroxisome bio-genesis, and endoplasmic reticulum-Golgi functions (Fig. 1A;see Table S1 in the supplemental material). Mutations affect-ing oxidative stress response (e.g., lys7�) and DNA damagerepair (e.g., rad50�) were also identified (Fig. 1B; see Table S1in the supplemental material), consistent with previous obser-vations that the drug causes oxidative damage (1, 20). Ahem14� mutant lacking the only nonessential gene of the hemebiosynthesis pathway in yeast was also sensitive to the drug(Fig. 1B; see Table S1 in the supplemental material). Amongall these mutants, those with mutations affecting multiple sub-units of the mitochondrial ATP synthase (e.g., Atp1p) andseveral other mitochondrial proteins, such as Yme1p, the cat-alytic subunit of the mitochondrial inner membrane i-AAAprotease involved in the turnover of unfolded or misfoldedmitochondrial membrane proteins (22, 26, 30, 38), exhibitedthe highest sensitivity (Fig. 1B and C; see Table S1 in thesupplemental material). The sampangine-hypersensitive phe-notype of the atp1� mutant was partly suppressed both byexogenously supplied ATP and by the antioxidant NAC (Fig.1C). These results together indicated that sampangine affectsmitochondrial functions, resulting in reduced energy and ele-vated ROS production. Interestingly, the sampangine-hyper-sensitive phenotype of the yme1� mutant was suppressed by

FIG. 1. Sampangine-hypersensitive mutants identified from genome-wide fitness profiling analysis. (A) Sampangine-hypersensitive mutantsdefine several highly enriched cellular components. In parentheses are the numbers of the Gene Ontology (GO) terms. ESCRT II, endosomalsorting complex required for transport II. (B) Representative mutants with differing sensitivities to sampangine (SMP). The ho� mutant servedas a surrogate wild-type (WT) control in this experiment and in experiments whose results are presented in all other figures. (C) Suppression ofsampangine hypersensitivity by exogenously supplied ATP (1 mM) and/or NAC (0.1 �g/ml) in atp1� and yme1� mutants.

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ATP but was barely suppressed by NAC under the same con-ditions (Fig. 1C), suggesting that the increase in ROS levelsmay not represent the most fundamental challenge faced bycells treated with sampangine.

The ATP synthase and Yme1p become essential when theETC or heme synthesis is defective. To identify the primaryfunction(s) affected by sampangine, we next performed ge-nome-wide synthetic lethality analyses with the atp1� andyme1� mutations using a high-throughput technology that wepreviously described (28, 29). Given that both mutations con-ferred hypersensitivity to sampangine yet the correspondingproteins seem to have distinct biological functions, we ex-pected that genome-wide synthetic lethality analyses with thesetwo mutations would identify common as well as distinct ge-netic interactions. Some of the common synthetically lethalinteractions would likely define the pathway(s) targeted bysampangine. Upon individual validation, we identified 172 and190 synthetically lethal or sick interactions for atp1� andyme1�, respectively, with the majority of them being surpris-ingly common (Fig. 2A). Most of these common interactionsaffected mitochondrial functions, particularly the mitochon-drial ribosome and the ETC (Fig. 2B and C; see Table S2 in thesupplemental material). While mutations of the mitochondrial

ribosome typically cause severe growth defects on their ownand tend to exhibit synthetic lethality interactions with othermutations, single mutations affecting mitochondrial ETC causeonly modest growth defects (data not shown), and their syn-thetic lethality interactions with atp1� and yme1� were thusdeemed more specific and significant. In addition, mutations ofmitochondrial ribosome could affect expression of some of theETC components. These results suggested that the atp1� andyme1� mutants both need a functional ETC to survive, and thiswas further corroborated by the observation that they wereboth highly sensitive to the ETC inhibitor antimycin (Fig. 2D).However, the ETC itself is unlikely a primary target of sam-pangine because it is dispensable for yeast cell survival,whereas the drug completely inhibits cell growth at �2 �g/ml.Instead, sampangine likely targets a process whose inhibitionseverely compromises the function of ETC.

We next considered the essential pathway(s) critical for thefunction(s) of ETC as a potential sampangine target(s) andfocused on heme biosynthesis for three main reasons. First, ourprevious studies have implicated heme in the antifungal activ-ity of sampangine. Second, heme is a physical component ofETC as a cofactor of the cytochromes. Third, synthetic lethalityinteractions were observed between heme deficiency (caused

FIG. 2. Sampangine inhibits heme synthesis and mitochondrial ETC. (A) Genome-wide synthetic lethality analyses with atp1� and yme1�identified a list of common mutations. The number of interactions for each is shown in parentheses. The genes are listed in Table S2 in thesupplemental material. (B) Common genetic interactions of atp1� and yme1� mostly affect mitochondrial protein synthesis and the respirationchain. (C) Synthetic lethality between atp1� and cox17� or hem14� and between yme1� and cox17� or hem14� revealed by tetrad analysis.(D) Hypersensitivity of the atp1� and yme1� mutants toward the ETC inhibitor antimycin (0.1 �g/ml). (E) Inability of wild-type yeast cells to useglycerol as the sole carbon source in the presence of sampangine (0.1 �g/ml). The cox17� and hem14� mutants were included as controls.



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by hem14� mutation) and both the atp1� and yme1� muta-tions (Fig. 2C; see Table S2 in the supplemental material).Inhibition of heme synthesis thus explains why the atp1� andyme1� mutants were sensitive to the drug. Consistent with themodel that sampangine inhibits the heme biosynthesis pathwayand subsequently causes defects in ETC, treating yeast with alow concentration of the drug blocked cell growth on glycerolas the sole carbon source. A similar blockade in cell growth onglycerol was observed with deletions in HEM14 or COX17,which encodes the copper metallochaperone required for theassembly of cytochrome c oxidase (3, 15) (Fig. 2E). Moreover,mutants lacking ETC components were no more sensitive tosampangine than a wild-type strain (Fig. 1B and data notshown), and a synthetic lethality interaction was not observedbetween hem14� and mutations of the ETC (cox17�) (Fig.3B), further indicating that sampangine treatment, hem14�,and cox17� all affect the same pathway.

The hem14� mutation phenotypically mimics sampanginetreatment. We further investigated whether the heme biosyn-thesis pathway represents a primary target of sampangine. Insuch a case, we expected that most if not all of the sampangine-sensitive mutants would exhibit lethality or a growth defectwhen heme synthesis is reduced. Consistent with this model,�58% (77 of 132) of the mutations that conferred sensitivitytoward sampangine (at 0.5 �g/ml) were indeed syntheticallylethal or caused sick interactions with the hem14� mutation(Fig. 3A; see Table S1 in the supplemental material). Theseincluded mutations affecting the ATP synthase, histone modi-fication, chromatin remodeling, peroxisome functions, oxida-tive stress response, and DNA repair (Fig. 3B; see Table S1 inthe supplemental material). For most of the mutations that didnot exhibit synthetically lethal or sick interactions withhem14�, the corresponding single mutants were also not sen-

sitive to a lower dose of the drug (0.2 �g/ml) (see Table S1 inthe supplemental material), reflecting the possibility that thedefect caused by a hem14� mutation was likely milder thanthat caused by treatment with sampangine at 0.5 �g/ml. Therest of the hem14� noninteractors likely either affect drugmetabolism or reflect additional functions targeted by sam-pangine that are not related to heme synthesis. In contrast,only �22% (29 of 132) of mutants with sampangine-sensitivemutations exhibited synthetic lethal or sick interactions withthe cox17� mutation, and all these were included within theinteraction profile of hem14� (Fig. 3A; see Table S1 in thesupplemental material). These results together indicated thatthe heme biosynthesis pathway is a primary target of sampang-ine and that the respiration defect represents only one of manypossible secondary effects of heme deficiency due to sampang-ine treatment.

The genetic interactions between a hem14� mutation andmutations affecting DNA repair, peroxisome biogenesis, andthe oxidative stress response (Fig. 3B) also suggested thatheme deficiency might lead to oxidative stress, possiblythrough crippling the ETC. Consistent with this model, thegrowth defect of the hem14� mutant was partly suppressed bythe antioxidant NAC (Fig. 3D). Thus, heme deficiency is atleast partly responsible for the increase in ROS levels observedin sampangine-treated cells (1, 20).

However, exogenously supplied hemin failed to completelyreverse the inhibitory effect of sampangine at relatively highconcentrations (Fig. 3D). The reason for this is currently notclear. Possibly, exogenous hemin is not readily taken up byyeast cells and/or efficiently transported to the right cellularcompartment (e.g., mitochondrion) once inside the cells. Thisis consistent with the observation that exogenous hemin onlyweakly suppressed the lethality caused by deleting components

FIG. 3. The heme biosynthesis pathway is a primary target of sampangine. (A) Venn diagram representation of genetic interaction profiles ofsampangine treatment (0.5 �g/ml) and a hem14� or cox17� mutation. The number of interactions for each is shown in parentheses. These resultswere derived from Table S1 in the supplemental material. (B) Synthetic sick interaction between hem14� and mutations affecting DNA repair(rad52�), peroxisome biogenesis (pex13�), and oxidative stress response (lys7�). Haploid-convertible heterozygous diploid deletion mutants thatharbored an hem14�::URA3 mutation and those of the indicated genotypes (kanMX as the marker) were sporulated, spotted on three haploidselection media, and evaluated for growth. The medium lacking both uracil and G418 allowed growth of the hem14�::URA3 mutant as well as thedouble mutant. The medium that lacked uracil but contained G418 allowed growth of double mutants only. The medium that contained both uraciland G418 allowed growth of the double mutants as well as single mutants of the indicated genotypes. (C) Suppressing the growth defect of ahem14� mutant with N-acetyl cysteine (0.1 �g/ml). (D) Exogenously supplied hemin (65 �g/ml) partly suppressed the lethality caused by deletingheme biosynthesis genes but failed to rescue the inhibitory effect of a lethal dose of sampangine (2 �g/ml).



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of the heme biosynthesis pathway even in the absence of sam-pangine (Fig. 3D). It is also possible that sampangine inhibitscell proliferation by targeting heme biosynthesis as well asother yet-to-be identified pathways. In this case, exogenouslysupplying hemin will unlikely restore all functions inhibited bysampangine.

Sampangine inhibits heme biosynthesis. Given the pheno-typic similarity caused by sampangine treatment and a hem14�mutation, as discussed above, we further tested the model inwhich sampangine inhibits heme biosynthesis. As expected, thehem14� mutant showed greatly reduced heme levels comparedto a wild-type strain (Fig. 4A). A similar effect was observedwhen a wild-type yeast strain was treated with sampangine atthe IC50 after one round of cell division (Fig. 4A). Such aninhibitory effect of sampangine on heme levels was also ob-served in human cells. Treatment with sampangine signifi-cantly reduced the intracellular levels of heme in two differenthuman cancer cell lines tested (Fig. 4B). These results togetherindicate that sampangine inhibits heme synthesis in both yeastand human cells.

We next investigated whether sampangine inhibits a specificstep in the heme biosynthesis pathway (Fig. 4C) (14). Wecompared the porphyrin intermediate metabolite profiles ofyeast cells grown in the presence and absence of the drug. Inparticular, we measured the levels of uroporphyrin III, copro-porphyrin III, and protoporphyrin IX, the oxidized intermedi-ate products of the fourth, fifth, and sixth steps of this pathway,respectively, because sampangine treatment leads to accumu-lation of a red pigmentation, which we thought to be porphyrin(1). Similar to what was reported previously (21), wild-typeyeast cells not treated with the drug mostly accumulated co-proporphyrin III and protoporphyrin IX, whereas the level ofuroporphyrin III was extremely low and beyond detection (Fig.4D). Treatment with the drug increased the total amount ofporphyrins by 5- to 20-fold in three independent experiments(data not shown). More importantly, the porphyrin profile inthe drug-treated cells was drastically altered, with the relativelevel of uroporphyrin greatly increased and the levels of co-proporphyrin III and protoporphyrin IX decreased (Fig. 4D).The levels of the hepta, hexa, and penta intermediates ofuroporphyrin III metabolism were also greatly increased (Fig.4D). These results were similar to those observed in mutantswith mutation of the uroporphyrinogen decarboxylase (UroD)encoded by HEM12 (21), suggesting that sampangine eitherinhibits Hem12 or hyperactivates enzymes in the precedingsteps, or both. Interestingly, sampangine treatment also mod-estly yet reproducibly caused reduced protein levels of Hem3and Hem4 and increased levels of Hem13 and Hem14 (Fig.4E), possibly due to feedback regulation of expression by theaccumulated intermediate metabolites. In contrast, the expres-sion levels of Hem12 itself and Hem1, Hem2, and Hem15,which lie farther away from Hem12 along the pathway, werenot significantly affected under similar conditions (Fig. 4E).

Sampangine likely hyperactivates Hem4 to inhibit hemesynthesis. We next tested the possibility that sampangine in-hibits UroD. Using a well-characterized in vitro assay with arecombinant human enzyme (32), we surprisingly found thatthe activity of purified UroD was not significantly affected byexcess sampangine at three concentrations tested (Fig. 5A). Onthe contrary, sampangine treatment significantly increased the

FIG. 4. Sampangine inhibits heme biosynthesis in vivo. (A) Sam-pangine treatment reduces heme levels in wild-type yeast. The hemeconcentration in the ho� mutant not treated with the drug, calculatedas fmol per �g of total protein, was set at 100%. Values shown aremeans � standard deviations from assays performed in triplicate. Theabsolute levels of heme in the hem14� mutant were close to thebackground levels and the detection limit of the assay. (B) Sampanginetreatments (0.01 �g/ml and 0.1 �g/ml) reduce heme levels in twohuman cancer cell lines, as indicated. The heme concentration in eachcell line not treated with the drug, calculated as fmol per �g of totalprotein, was set at 100%. Values shown are means � standard devia-tions from assays performed in triplicate. (C) Heme biosynthesis path-way in yeast. CoA, coenzyme A. (D) Effects of sampangine treatmenton porphyrin profiles in a wild-type yeast strain. Uro, uroporphyrin III;Hepta, heptaporphyrin III; Hexa, hexaporphyrin III; Penta, pentapor-phyrin III; Copro, coproporphyrin III; Proto, protoporphyrin IX. Val-ues shown are means � standard deviations from assays performed intriplicate. (E) Effects of sampangine treatment (1 �g/ml) on proteinlevels of each of the eight enzymes of the yeast heme biosynthesispathway analyzed by Western blotting. Enzymes were expressed astandem affinity purification (TAP) tag fusion proteins from the endog-enous loci. The fusion proteins are indicated with black arrows. Tub2was used as a loading control.



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activities of both porphobilinogen deaminase (PBGD) (P �0.0009) and uroporphyrinogen III synthase (UIIIS) (P � 0.01),enzymes preceding UroD in the heme biosynthesis pathway(Fig. 5A). That sampangine stimulates the activities of thesetwo enzymes could explain the increased levels of uroporphy-rinogen III observed in drug-treated yeast cells (Fig. 4D).However, it was not clear how this might be related to theblockade of heme synthesis by sampangine treatment observedin vivo (Fig. 4A). It is possible that hyperactivation of PBGDand/or UIIIS disrupts the balance within the entire heme syn-thesis pathway and leads to decreased heme levels. To test thishypothesis, we measured cellular levels of hemin in yeaststrains that overexpress HEM3, HEM4, and HEM12, genesencoding PBGD, UIIIS, and UroD, respectively (2, 10, 18),from high-copy-number plasmids. Overexpressing both HEM3and HEM12 increased heme levels in yeast cells, and theseeffects were blocked by sampangine treatment (Fig. 5B). Onthe contrary, HEM4 overexpression reproducibly caused a re-duction in heme levels even in the absence of sampangine (P �0.005) (Fig. 5B). Consistent with this observation, overexpres-sion of HEM4 caused a growth defect in both a wild-type strainand an atp1� mutant (Fig. 5C). This inhibitory effect wasfurther exacerbated by sampangine treatment (Fig. 5C). Theseresults together suggested that a higher level of activity ofHem4 due to sampangine treatment or genetic overexpressioninhibits heme biosynthesis. However, reducing the dosage of

HEM4 in a diploid yeast strain by half did not confer sampang-ine resistance (data not shown). An attempt with targetedrandom mutagenesis also failed to produce sampangine-resis-tant mutants, despite screening about 50,000 distinct alleles ofthe HEM4 gene (data not shown), possibly because potentialresistance alleles are also nonfunctional.

DISCUSSION

By using a combination of genomic, genetic, and biochemi-cal approaches, we have demonstrated that the plant alkaloidsampangine antagonizes cellular proliferation mainly by inhib-iting heme biosynthesis, a function essential for cell survival.This mechanism of action is apparently conserved in yeast andhuman cells, further proving yeast to be a valuable system forinvestigating the mechanisms of action exhibited by bioactivesmall molecules. In order to discover the pathway(s) mostsignificantly affected by sampangine treatment, we used a com-bination of genome-wide deletion mutant fitness profiling andsynthetic lethality analysis. We first identified mutations thatconfer the highest sensitivity toward the drug. Cells with rep-resentative mutations were then used in genome-wide syn-thetic lethality analyses to reveal the cellular functions or path-ways most likely affected by the drug. This approach will likelybe useful in revealing the mechanisms of action exhibited byother drugs. Apparently, this was not the most straightforwardapproach for identifying drug targets in yeast cells. However, itwas necessary with sampangine because both screening thegenome-wide heterozygous diploid deletion mutants for drug-induced haploinsufficiency and analyzing a genome-wide geneoverexpression library for drug-resistant clones, which couldpresumably directly reveal drug targets (6, 12, 23), had failed.We note that some heterozygous diploid mutants of the hemesynthesis pathway were comparatively more sensitive to thedrug than a wild-type strain (1) (data not shown). However,their defects were too subtle to be detected by the genome-wide screen that we performed.

Our results suggest that heme biosynthesis is a primary tar-get of sampangine in yeast cells and likely also in human cells.This was supported by the evidence that heme deficiency dueto a hem14� mutation largely mimicked the effects of sam-pangine treatment and that sampangine treatment inhibitedheme synthesis in vivo. We also showed that heme deficiency isat least partly responsible for the increased levels of ROSobserved in sampangine-treated cells (1, 20), possibly due to adefect in the ETC. In particular, we observed synthetic sickinteractions between heme deficiency (due to the hem14� mu-tation) and oxidative stress response defects (due to the lys7�mutation) (Fig. 3C). In addition, the growth defect of ahem14� mutant was partly suppressed by the antioxidant NAC(Fig. 3C). Both sampangine treatment and heme deficiencywere also shown to downregulate expression of multiple genesinvolved in iron uptake in yeast (1, 9). This could deprive cellsof iron and further contribute to heme deficiency. This alsopoints to the possibility that heme deficiency might also beresponsible for the iron depletion observed in mycobacteriatreated with the structurally related ascididemin (5). Takentogether, inhibition of heme biosynthesis is likely the primarycause of the various biological effects of sampangine and itsanalogs.

FIG. 5. Sampangine likely hyperactivates uroporphyrinogen IIIsynthase to inhibit heme synthesis. (A) Effects of sampangine on therelative activities of uroporphyrinogen III synthase, porphobilinogendeaminase, and uroporphyrinogen decarboxylase in vitro. The activityof each enzyme in the absence of the drug was set at 100%. Valuesshown are means � standard deviations from assays performed intriplicate. (B) Effects of overexpressing HEM3, HEM4, or HEM12 oncellular heme levels in the presence or absence of sampangine (1�g/ml). Values shown are means � standard deviations from assaysperformed in triplicate. (C) HEM4 overexpression inhibits yeastgrowth and causes sampangine hypersensitivity. Sampangine was usedat 1 �g/ml and 0.05 �g/ml for the ho� and atp1� mutants, respectively.



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Our results have also provided a glimpse into how sampang-ine might inhibit heme biosynthesis. It most likely involveshyperactivation of UIIIS (encoded by HEM4), which is by farthe most active enzyme within the heme biosynthesis pathway(14). This model is based on the observations that sampanginestimulates the activity of human UIIIS in vitro and that over-expressing Hem4 inhibits heme synthesis and causes growthdefects in yeast cells (Fig. 5). In addition, yeast cells treatedwith sampangine also accumulate higher levels of uroporphy-rinogen III, the product of UIIIS, and its decarboxylation de-rivatives (Fig. 4A). However, it still remains to be investigatedhow sampangine treatment and hyperactivation of UIIIS mightinhibit heme synthesis. It certainly does not involve reducedlevels of expression of any of the downstream enzymes of thepathway (Fig. 4E). Overexpressing none of the downstreamenzymes suppressed the detrimental effects of HEM4 overex-pression and sampangine treatment (data not shown). Wespeculate that Hem4 hyperactivation might somehow causeirreversible damage to the pathway. This might help to explainthe puzzling observation that heterozygous diploid mutantswith mutations of Hem1 and Hem2, which lie upstream toHem4 in the heme synthesis pathway, are hypersensitive tosampangine (1). Possibly, reducing the gene dosage of theupstream components in these mutants has little effect onHem4 activation by the drug. However, once Hem4 is hyper-activated and the pathway is damaged, reducing the flux of thepathway further exacerbates the defects in heme synthesis.

Given that sampangine inhibits heme biosynthesis in bothyeast and human cells and possibly other organisms, it mightlack the needed specificity as a therapeutic agent against hu-man pathogens and cancer cells. However, high specificitycould still be achieved by exploiting unique genetic interactionnetworks within particular pathogens or cancer cell types. Thelist of sampangine-hypersensitive mutations identified in thisstudy could aid in identifying pathogens or human cancer typesthat might be particularly sensitive to this drug and be a guidefor selecting drug combinations that exhibit higher therapeuticpotency and/or broader therapeutic indexes. Regardless ofthese, sampangine could serve as a chemical tool for investi-gating the physiological functions of heme metabolism in var-ious biological systems and for studying the molecular mech-anisms of human diseases related to heme deficiency, becauseits effect in yeast was largely mimicked by that of heme defi-ciency (Fig. 3A; see Table S1 in the supplemental material).However, for such applications, one should take into consid-eration possible off-target effects of sampangine because hemedeficiency is probably not the only biological effect of sampang-ine treatment in yeast. This is because exogenous hemin partlyrescued the growth inhibition caused by deleting heme biosyn-thesis genes yet completely failed to suppress sampangine’slethal effects at high concentrations (Fig. 3D). Possibly, inhi-bition of the heme biosynthesis pathway by sampangine con-fers additional cellular defects that cannot be overcome simplyby supplying cells with heme. For example, the accumulation ofuroporphyrinogen III and potentially increased levels of othermetabolic products derived from uroporhyrinogen III mightalso contribute to sampangine’s cytotoxic effect. It is also pos-sible that, in addition to heme biosynthesis, sampangine di-rectly inhibits an additional yet-to-be discovered pathway.

Our study also revealed a surprising synthetic lethality inter-

action relationship between mutations in the mitochondrialATP synthase and those in the ETC (Fig. 2B and C; see TableS2 in the supplemental material). Mitochondrial ATP synthaseand ETC are together required for producing ATP via respi-ration, which is thought to be dispensable for yeast cell survivalunder normal growth conditions because this organism getsenough energy from glycolysis alone. We also found that thelethality of an atp1� cyc3� double mutant was not suppressedby exogenously supplied ATP (data not shown), even thoughthe same amount of ATP partly suppressed the sampanginehypersensitivity of an atp1� mutant (Fig. 1D). NAC, whenused alone or together with ATP, also failed to restore growthin the atp1� cyc3� double mutant (data not shown), suggestingthat the lethality of this mutant simply due to decreased ATPproduction and increased ROS levels is unlikely. Therefore, inaddition to ATP production, mitochondrial ATP synthase andthe ETC together likely play an additional undefined role thatis essential for yeast cell survival. Understanding the molecularmechanism of the lethality observed in the atp1� cyc3� doublemutant could provide insights into such a novel function ofmitochondrial ETC and ATP synthase.

ACKNOWLEDGMENTS

We thank Ping Shi for providing human cancer cell lines and advicefor measuring heme in human cells. We also thank Hector Bergoniaand Isiah Davies for their assistance in porphyrin separation andmethod development.

This work was supported by NIH grants R01 HG004840 (to X.P.),R01 DK020503 and U54 DK083907 (to J.D.P.), and R01 AI27094 (toA.M.C.) and by USDA-ARS Specific Cooperative Agreement 58-6408-2-0009 (to the University of Mississippi).

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