methylthioadenosine phosphorylase regulates … · the gene encoding methylthioadenosine...

Methylthioadenosine Phosphorylase Regulates Ornithine Decarboxylase by

Production of Downstream Metabolites*

Ahmad L. Subhi‡, Paula Diegelman§, Carl W. Porter§, Baiqing Tang‡, Zichun J. Lu¶,

George D. Markham¶, and Warren D. Kruger‡||

From the Divisions of ‡Population Science and ¶Basic Science, Fox Chase Cancer Center,

Philadelphia, PA 19111 and the §Roswell Park Cancer Institute, Pharmacology and

Therapeutics Department, Buffalo, NY 14263

* This work was supported by United States Army Grant DAMD17-97-1-7707, USPHS grant

CA-22153 and Core grant CA-06927 from the National Institutes of Health and by an

appropriation from the Commonwealth of Pennsylvania. We also acknowledge the work of the

Sequencing and Cell Culture Facilities at Fox Chase Cancer Center.

||To whom correspondence should be addressed: Fox Chase Cancer Center, 333 Cottman

Avenue, Philadelphia, PA 19111-2497. Tel: 215-728-3030; Fax: 215-214-1623;

E-mail: [email protected]

Running title: MTAP regulation of ODC

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 23, 2003 as Manuscript M308451200 by guest on June 7, 2018

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The abbreviations used are: MTA, methylthioadenosine; MTR1P, methylthioribose-1-

phosphate; MTAP, methylthioadenosine phosphorylase; ODC, ornithine decarboxylase ;

DFMO, α-difluoromethylornithine; SAM, S-adenosylmethionine, dSAM, decarboxylated S-

adenosylmethionine.

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SummaryThe gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the

methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor

suppressor gene in cell culture (1-6). Overexpression of the polyamine biosynthetic enzyme

ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be

tumorigenic in vitro and in vivo (7-9). In this paper, we demonstrate a novel regulatory pathway

in which the methionine salvage pathway products inhibit ODC activity. We show that in

Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1∆ cells have an eight-

fold increase in ODC activity resulting in large elevations in polyamine pools. Mutations in

putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and

elevated polyamines. Addition of the penultimate salvage pathway compound 4-methylthio-2-

oxobutanoic acid (MTOB) represses ODC levels in both MTAP deleted yeast and human tumor

cell lines, indicating that MTOB acts as a negative regulator of polyamine biosynthesis.

Expression of MTAP in MTAP deleted MCF-7 breast adenocarcinoma cells results in a

significant reduction of ODC activity and reduction in polyamine levels. Taken together, our

results show that products of the methionine salvage pathway regulate polyamine biosynthesis,

and suggest that MTAP deletion may lead to ODC activation in human tumors.

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INTRODUCTION

A quarter century ago, Toohey first recognized that certain murine malignant hematopoietic

cell lines lacked methylthioadenosine phosphorylase (MTAP) activity (10). MTAP is a key

enzyme in the methionine salvage pathway (see Figure 1). This pathway functions to salvage

methylthioadenosine (MTA), which is formed as a byproduct of polyamine metabolism.

Phosphorolysis of MTA by MTAP results in the conversion of MTA into adenine and

methylthioribose-1-phosphate (MTR1P). A series of reactions then salvages the methyl-thio

group from MTR1P to form methionine. This pathway has been most extensively studied in

Klebsiella pneumoniae (11-14), but has also been shown to exist in rat liver (15-17) and in

Saccharomyces cerevisiae (18,19).

Loss of MTAP activity through gene deletion is common in many kinds of human cancers

including non-small cell lung cancer, glioma, T-cell acute leukemia, bladder cancer,

osteosarcoma and endometrial cancer (3,5,20-22). Recently, we found that expression of MTAP

in a MTAP-deleted breast adenocarcinoma cell line (MCF-7) resulted in a dramatic inhibition

of tumorigenicity in vitro and in vivo, showing that MTAP can function as a tumor suppressor

gene (1). We also found that MTAP expression causes a significant decrease in intracellular

polyamine levels and alters the ratio of putrescine to total polyamines. Consistent with this

observation, the polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) inhibits

the ability of MTAP deficient MCF-7 cells to form colonies in soft-agar, while addition of the

polyamine putrescine stimulates colony formation in MTAP expressing MCF-7 cells. These

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results indicate that MTAP’s tumor suppressor activity may be mediated by its effect on the

intracellular polyamine pools.

Polyamines are small, aliphatic amines involved in a variety of cellular processes including

transcription and apoptosis (8). The rate-limiting enzyme in the production of polyamines is

ornithine decarboxylase (ODC). Elevated ODC activity has been observed in a wide variety of

human and animal tumors, and overexpression of ODC in NIH/3T3 cells is sufficient to cause

transformation in vitro (7,23). Transgenic mice overexpressing ODC in skin develop skin

tumors at a high frequency (24). These observations show that overexpression of ODC is

tumorigenic.

Examination of the S. cerevisiae genome for MTAP homologues suggests that the MEU1

gene, with 35% identity and 53% similarity over 275 amino acids, may encode the yeast MTAP

homologue. Furthermore, recent studies show that cells lacking MEU1, in combination with a

mutation that allows yeast to take up methylthioadenosine, are unable to grow on medium

containing MTA as the sole sulfur source (25). MEU1 was initially identified in a screen for

genes that regulate expression of the yeast ADH2 gene (26). Overexpression of MEU1 increased

ADH2 expression while deletion of MEU1 resulted in reduced ADH2 expression. At the time

these experiments were published the MTAP gene had not yet been identified, so the relationship

between MEU1 and MTAP was unknown. These experiments demonstrate that MEU1 regulates

ADH2 expression and indicate a link between MEU1 and gene regulation.

In this paper we show that MEU1 encodes yeast MTAP, and we characterize the effect of

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loss of MTAP in Saccharomyces cerevisiae and human tumor cells. Our findings show that there

is a regulatory link between the methionine salvage pathway and the polyamine pathway.

Specifically, the penultimate metabolite, 4-methylthio-2-oxobutanoic acid (MTOB), acts to

repress ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. These

results explain why MTAP deletion causes altered polyamine profiles, and suggest that MTAP’s

tumor suppressor activity may be due to its effect on ODC activity.

EXPERIMENTAL PROCEDURES

Plasmid pMEU1 construction – The yeast MEU1 ORF was PCR amplified from total yeast

DNA using primers MEU1-1F (5′- TCT GTG AAA CAT GTC) and MEU1-1R (5′ CAG TCC

CCA AGG GGG). The resulting 1.3 Kb product was the cloned into pCR2.1 (Invitrogen) and

designated pCR2.1::MEU1. PCR2.1::MEU1 was digested with BamH1 and NotI and the

resulting 1.3 Kb insert was cloned into pRS316 (27) digested with the same enzymes.

Yeast Strains – Yeast strains used are indicated in Table 1. Isogenic MEU1 and meu1∆ strains

were created by transformation of AS3-2a (a ura3∆ met15∆ meu1∆::LEU2 leu2∆) with pMEU1 or

empty vector pRS316. All other strains used were created by the S. cerevisiae deletion project (28).

Standard yeast growth media was used as described by Sherman (29). Polyamine free media was

prepared as described by Balasundaram (30).

Mammalian Cells – MTAP expressing and mutant expressing MCF-7 cells were created and

grown as previously described (1). MIA PaCa-2 pancreatic adenocarcinoma cells were obtained

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from the ATCC and grown as previously described (31).

Yeast Growth Studies – Yeast cells were grown to stationary phase and then diluted to an

OD600 of 0.05 in the indicated media. Cells were then grown in liquid media with shaking at 30oC

for 48 hours, at which time OD was determined.

Extract preparation – Yeast cultures (45 ml) were grown aerobically to an OD600 of 0.5-1.0,

pelleted, washed with 0.9ml of lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1 mM PMSF),

and resuspended in 750µl lysis buffer. Glass beads (0.5 mm, one-half weight of the cell pellet)

were then added to make up approximately 50-70% of total volume. Cells were lysed by using a

BeadBeater at 4°C with maximum speed (4500 rpm) for 1 minute three times and kept on ice

between pulses (Biospec Products). After lysis, the tubes were centrifuged for 4 minutes and the

supernatant was transferred to a new tube and glycerol was added to a final concentration of

15%. The protein concentration of yeast extracts was determined by the Coomassie Blue protein

assay reagent (Pierce) using bovine serum.

Mammalian cell lysates were prepared by 3 cycles of freeze-thawing at –80°C in 20 mM

KH2PO4, pH 7.4 containing 1 mM phenylmethylsulfonyl fluoride (Sigma) and 1 mM dithiothreitol

(Sigma).

Western blot analysis Forty µg of extract were run on pre-made 7% NuPage Tris-acetate

SDS gels (Invitrogen) and electrotransferred to PVDF membrane (Biorad) using a standard

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protocol (32). Samples were probed with a 1/10,000 dilution of yeast anti-ornithine

decarboxylase antiserum raised in rabbits (a generous gift from Martin Hoyt and Phil Cofino,

UCSF). Secondary antibody anti-rabbit HRP linked antibody was obtained from Amersham

Biosciences and used at a 1/3,000 dilution. The antibody-Antigen complex was visualized on

photographic film after treatment with SuperSignal West Dura Extended Duration Substrate

(Pierce biotechnology).

Measurement of ornithine decarboxylase, MTAP, and AdoMet decarboxylase activity –

Ornithine decarboxylase activity was assayed by measuring the 14CO2 formed by

decarboxylation of [1-14C] labeled ornithine in as previously described (33). L-carboxy 14C

ornithine (5mCi/mmol) was obtained from Moravek Biochemical (Brea, CA). 20 µg of yeast or

mammalian extract was used per reaction. Reactions were performed between three and seven

times per sample and results are presented as the average with the standard error. Adomet

decarboxylase activity was assayed by measuring the amount of 14CO2 released by

decarboxylation of [1-14C] labeled AdoMet as described (34).

MTAP activity in yeast extracts was determined using a spectrophotometric assay as

described previously (1). One unit of MTAP activity is defined as the amount that catalyzes the

formation of 1µmol of adenine/min.

Measurement of intracellular polyamine levels by using HPLC – Yeast cells grown in

synthetic complete media were collected, protein extracts were prepared, and then 100µl

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supernatant added to 100µl 0.6N PCA and allowed to sit on ice for 5 minutes. The sample was

centrifuged at 15,000g for 10 min. 100µl of supernatant was dansylated and analyzed by HPLC

as described (35).

RESULTS

MEU1 encodes MTAP in S. cerevisiae – Early studies showed that yeast extracts contain

MTAP activity, but did not identify the gene encoding the activity (18,19). A BLAST search of

the S. cerevisiae genome with human MTAP (NP_002442) reveals two open reading frames,

YLR209C and YLR017W, with significant sequence similarity. Of the two, YLR017W (also

known as MEU1) has the most similarity with 35% identity over 275 amino acids to human

MTAP. To test the possibility that MEU1 encoded MTAP, we constructed isogenic MEU1 and

meu1∆ strains and measured MTAP activity in whole cell extracts (see Materials and Methods).

We found that MEU1 yeast extracts contained 0.38 units of MTAP activity, while extracts from

meu1∆ strain had no detectable activity (<0.01units). A second experiment to confirm our

hypothesis involved examining the growth of isogenic meu1∆ and MEU1 strains with

auxotrophic markers for adenine and methionine in media in which MTA had been substituted

for methionine and adenine. We found that wild-type but not meu1∆ yeast can grow using

methylthioadenosine (MTA) as a substitute for methionine and adenine (Figure 2). Taken

together, these observations show that MEU1 encodes yeast MTAP.

Effect of meu1∆ on polyamine pools and ODC activity Since MTA is a by-product of

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polyamine production, we suspected that cells lacking MTAP might have altered polyamine

pools due to end-product inhibition. Therefore, we measured the intracellular level of

polyamines (putrescine, spermine and spermidine) in an isogenic wild-type strain and in a

meu1∆ strains using HPLC (see Materials and Methods). We found that in synthetic complete media,

MEU1 cells had undetectable levels of putrescine, while meu1∆ cells contained at least a 20-fold

increase in putrescine (Figure 3a). Surprisingly, we also found that spermidine levels were

significantly increased and that the total polyamine content of meu1∆ cells was more than twice

that of isogenic wild-type yeast (62 vs. 28 nmoles/mg).

To explain the overall elevation in polyamine levels we examined ornithine decarboxylase

(ODC) activity in wild type and meu1∆ yeast. ODC is the rate-limiting enzyme in polyamine

production and is regulated at the post-transcriptional level (36). We found that cell extracts

from meu1∆ yeast had an eight-fold increase in ODC activity compared to isogenic wild-type

yeast (Figure 3b). This difference appeared to be due to elevated levels of ODC protein. We

were unable to detect ODC protein by immunoblot in extracts derived from MEU1 cells as has

been previously observed (37) and from ODC deficient cells (spe1∆), but we saw substantial

expression in meu1∆ extracts (Figure 4a, lanes 1-3). The increase in protein levels appeared to

be even greater then the measured activity, suggesting that not all of the protein was active (lanes

1 and 2). We also noticed that meu1∆ yeast formed significantly larger colonies compared to

isogenic MEU1 yeast on polyamine free media, suggesting that the elevated ODC is important

for growth under theses conditions (Figure 4b). These results show that MEU1 represses ODC

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expression and activity.

We also examined the effect of MEU1 on S-adenosylmethionine decarboxylase (SAMDC)

activity. We found that extracts from meu1∆ cells had twice the SAMDC activity of isogenic

MEU1 (7.7 ± 0.3 vs. 14.6 ± 0.8 nmol/mg/hr). This result is consistent with the idea that MEU1

acts to repress polyamine synthesis.

SPE2 and MEU1 regulate ODC through different mechanisms – Previous work has shown

that mutations in SPE2, encoding SAM decarboxylase, also cause a dramatic increase in ODC

protein levels (36) (also Figure 3a, lane 4). It was also shown that the ODC overexpression

observed in a spe2 mutant could be suppressed by the addition to spermidine to the media. We

compared ODC expression in spe2∆ and meu1∆ strains in the presence and absence of

spermidine. Meu1∆, spe2∆ and meu1∆/spe2∆ double mutants were grown in polyamine free

medium with or without the addition of exogenous spermidine. When we measured ODC

enzyme activity we found that addition of 0.1 mM spermidine to the media caused a 416%

decrease in ODC activity in spe2∆ cells, but only a 15% decrease in meu1∆ cells (Figure 5a).

The double mutant strain had an intermediate phenotype, with a 52% decrease. Differential

response to spermidine was also observed at the protein level as shown by immunoblot analysis

(Figure 5b). In the spe2∆ strain addition of spermidine resulted in undetectable levels of ODC,

while it had no detectable effect on meu1∆ strain. These results show that elevation of ODC

levels in meu1∆ strains and in spe2∆ strains are due to different regulatory systems.

MTA has no effect on ODC protein levels MTA, the substrate of MTAP, is known to be a

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strong competitive inhibitor of spermine synthase, SAH hydrolase, and other methyltransferases

(38-40). We tested the possibility that elevated MTA levels could cause the elevation of ODC

levels we observed in meu1∆ yeast. We grew cultures of MEU1, meu1∆, and spe2∆ yeast with

and without added 5 mM MTA to media, and examined ODC protein by immunoblot (Figure

5c). We saw no significant increase in ODC levels of any of the three strains by the addition of

exogenous MTA. This result indicates that build-up of MTA is not responsible for the elevated

ODC levels observed in meu1∆ yeast.

Mutations in E1 and E2 homologues effect ODC and polyamine levels – Given the result above,

we reasoned that a salvage pathway metabolite downstream of MTA might play a key role in

repressing ODC levels. To test this hypothesis, we examined ODC levels in yeast strains deleted

for genes similar to two known salvage pathway enzymes from Klebsiella oxytoca. The E1 protein

from Klebsiella catalyzes the conversion of 2,3-diketo-5-methylthio-1-phosphopentene to 1,2-

dihydroxy-3-keto-5-methylthiopentene, while the E2 protein catalyzes the subsequent formation

of 2-keto-4-methylthiobutyrate (see Figure 1)(6,41-43). We searched the S. cerevisiae genome

for ORFs similar to these proteins and found one for each gene. Yeast YEL038W encodes a protein

that is 37% identical to Klebsiella E1, while yeast YMR009W encodes a protein with 26% identity

to E2. We obtained deletions of each of these genes from the Saccharomyces Genome Deletion

Project (28). Deletion strains for each of these proteins were viable and whole cell extracts were

prepared and examined for polyamine levels, ODC activity, and protein levels. E1 and E2 deletion

strains had elevated ODC activity and protein levels compared to the MEU1 control strain (see

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Figure 3b and Figure 4a). However, neither strain had quite the same level of induction as observed

in the meu1∆ strain. The polyamine profiles also showed an intermediate effect (Figure 3a).

Spermidine levels were clearly elevated in the E1 and E2 mutants, but putrescine was still below the

limit of our detection. These results show that mutations in enzymes downstream of MTAP cause

elevated ODC levels and elevated levels of spermidine.

MTOB repression of ODC in meu1∆ Based on the findings above and our knowledge of the

biochemical pathway, we reasoned that perhaps MTOB was a negative regulator of ODC. We

tested this hypothesis by growing the various mutants in media in which 1 mM MTOB was

substituted for methionine. Yeast grown in MTOB had identical doubling times as yeast grown in

methionine (data not shown). However, immunoblot analysis of ODC clearly shows that the

addition of MTOB dramatically abolishes the elevated ODC levels observed in meu1, E1, and E2

mutants (Figure 6). This finding shows that MTOB represses ODC protein levels.

MTAP and MTOB repress ODC in human tumor cells – Previously we have described the

creation of isogenic MTAP positive and MTAP negative MCF-7 breast adenocarcinoma cell lines,

and showed that MTAP caused a three-fold decrease in total polyamine levels (see Table 3, ref 1).

To see if this repression might be related to ODC repression, we measured ODC activity in two

MTAP expressing and two MTAP non-expressing clones (Figure 7a). Depending on the clones

compared, there is a three to nine-fold drop in ODC activity in the MTAP expressing compared to

the non-expressing cells. These results show that MTAP represses ODC in mammalian cells as

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well as yeast.

We also examined if exposure to MTOB could repress ODC activity in mammalian cells. For

these experiments we used MCF-7 cells and MIAPaca-2 cells, a pancreatic adenocarcinoma cell

line deleted for MTAP (31). Cells were grown in regular methionine containing media and then

placed in either media with methionine or lacking methionine and supplemented with either 0.2 or 2

mM MTOB. Previously we have shown that MCF-7 cells can utilize MTOB as a source of fixed

sulfur and have identical doubling times on either methionine or MTOB (44). We found similar

growth results with MIAPaca-2 cells (data not shown). After twenty-four hours cells were

harvested and ODC activity was determined (Figure 7b). Cells from both lines treated with either

concentration of MTOB had significant decreases in ODC activity compared to cells growing in 100

µM methionine. These results indicate that MTOB acts as a negative regulator of ODC activity in

mammalian cells.

DISCUSSION

In this report we demonstrate that the deletion of MTAP causes activation of ODC resulting in

elevated polyamine pools. Our data indicates that a specific downstream salvage pathway

metabolite, MTOB, may be a key player in this process. In yeast, mutations in putative salvage

pathway genes located between MTAP and MTOB caused elevated ODC protein and activity

levels. In both MTAP-deleted yeast and human cells, addition of MTOB to the media causes a

substantial reduction in ODC activity. Our studies cannot determine if MTOB is a direct regulator

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of ODC or whether some metabolite of MTOB is involved. Studies using BAF3 murine lymphoid

cells have demonstrated that MTOB can be converted to malondialdehyde and methional in cell

extracts (45). Interestingly, MTOB and methional at high concentrations can induce apoptosis in

BAF3 cells. It has been shown that drugs that cause depletion of polyamines can sometimes also

cause apoptosis in tumor derived cell lines (46). Thus it is possible that in BAF3 cells, MTOB may

cause apoptosis by depletion of polyamine pools.

We found in S. cerevisiae that mutations in the homologues from Klebsiella E1 and E2 had

more modest effects on ODC activation than MTAP deletion. If MTOB were the only inhibitor of

ODC, one would expect levels to be identical. There are two possible explanations. First, it may be

that there are alternate enzymatic pathways in yeast that can convert methylthioribose-1-phosphate

to MTOB, and thus the E1 and E2 mutants may have some low level of MTOB present. Consistent

with this idea, Dibner et al. reported that methylthioribose-1-phosphate could be converted to 2-

hydroxy-4-methylthiobutanoic acid which could then be converted to MTOB (47). Alternatively,

other metabolites in the pathway may also inhibit ODC levels. For example, MTOB and

methylthioribose-1-phosphate may both be repressors of ODC.

In this paper we show several lines of evidence that the yeast MEU1 gene encodes MTAP,

including amino acid similarity, requirement of MEU1 for enzyme activity, and growth on MTA

containing media. The MEU1 gene was initially identified in a genetic screen designed to isolate

mutations affecting transcriptional regulation of ADH2 (26). Why did MTAP come up in such a

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screen? Our finding that MTAP deletion alters polyamine homeostasis by overexpression of ODC

suggests a possible explanation. Recent evidence indicates that there is a significant link between

polyamines, enzymes involved in chromatin remodeling, and transcriptional regulation. In yeast it

has been found that mutations that reduce polyamine levels are able to bypass the need for the core

histone deacetylase, GCN5, for the expression of certain genes (48). In ODC over-expressing

transgenic mice both intrinsic histone acetyltransferase (HAT) and deacetylase activities (HDAC )

are elevated (49,50). Alterations in HAT and HDAC activities would be expected to have effects on

a large number of genes (51). Consistent with this hypothesis, microarray analysis of 15,000 ESTs

in isogenic MTAP+ and MTAP- MCF-7 cells indicate that over 200 genes are either two-fold

induced or repressed by MTAP expression (B. Tang and W.D. Kruger, unpublished data).

ODC levels in yeast and mammalian cells are regulated post-transcriptionally. In higher cells,

the antizyme protein targets ODC for degradation by the proteosome (52). Antizyme production

requires a frame-shifting event that is greatly stimulated when polyamines are absent. S. cerevisiae

lacks a canonical antizyme, but appears to have a similar post-transcriptional mechanism for ODC

regulation in response to polyamine depletion (36). Our work here shows that the salvage pathway

products also negatively regulate ODC. Thus ODC is feed-back regulated by both of its

downstream pathways. We did not observe a “hyper” increase in the ODC levels in the double

mutant suggesting that both feedback processes may be affecting the same underlying mechanism,

i.e. disruption of either feedback loop results in elevated ODC.

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Our findings also support our earlier hypothesis that MTAP acts as a tumor suppressor by

altering polyamine pools. Previously, our lab has shown that expression of MTAP in MTAP

deleted MCF-7 cells suppresses anchorage-independent growth, tumor formation in SCID mice,

and causes significant reduction in polyamine levels (1). This reduction in polyamines is easily

explained by inhibition of ODC by MTAP. We also demonstrated in our previous work that

addition of putrescine could partially restore anchorage-independent growth to MTAP expressing

cells, indicating that at least part of MTAP’s ability to suppress tumorigenicity is due to its affect on

polyamine production. However, it is possible that MTAP deletion in tumor cells may be affecting

other pathways in addition to polyamine biosynthesis that contribute to tumorigenicity. Future

experiments will focus on answering this question.

Acknowledgements – We thank Martin Hoyt and Phil Cofino for the use of their yeast ODC

anti-serum. We also thank Randy Strich, Elizabeth Henske, and Eric Moss for their critical reading

of the manuscript.

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Table 1. Strain List

Strain Relevant Genotype Source

AS3-

2a(pMEU1)

a ura3∆ met15∆ meu1∆::LEU2

leu2∆ pMEU1

WY52-5A1 X YLR209C

(This study)

AS3-

2a(pRS316)

a meu1∆::LEU2 leu2∆ ura3∆

met15∆ pRS316

WY52-5A1 X YLR209C

(This study)

YOL052C a spe2∆::kanMX met15∆ Saccharomyces Genome

Deletion Project

YKL184W a spe1∆::kanMX met15∆ Saccharomyces Genome

Deletion Project

AY32 α spe2∆::kanMX meu1::LEU2

met15∆

YOL052CX WY525A1

(This study)

YEL038W (E1) a YEL038∆::kanMX met15 ∆ Saccharomyces Genome

Deletion Project

YMR009W (E2) a YMR009∆::kanMX met15∆ Saccharomyces Genome

Deletion Project

WY52-5A1 α met6∆ meu1∆::LEU2 ura3∆

trp1∆ leu2∆

WK10-2c (Kruger lab) X

MC71-18b

AS24-1b

(pMEU1)

a ura3∆ met15∆ meu1∆::LEU2

leu2∆ pMEU1 ade2

WY131xAS3-2a

AS24-1b

(pRS316)

a meu1∆::LEU2 leu2∆ ura3∆

met15∆ ade2(pRS316)

WY131xAS3-2a

YLR209C a his3∆ leu2∆ met15∆

pnp1∆::kanMX ura3∆

Saccharomyces Genome

Deletion Project

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WY131

(ATCC 200869)

α ade2 leu2∆ lys2∆ met15∆ ura3∆ ATCC (Isogenic to strains used

in Genome Deletion Project

see (53)

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Figure Legends

Figure 1. Methionine salvage and polyamine pathways. Enzymes are shown in italics. Yeast

genes are shown in parenthesis.

Figure 2. Growth of MTAP+ and MTAP- yeast cells on MTA. Yeast strains AS24-1b (MEU1)

and AS24-1b (meu1∆) were diluted to OD600=0.05 in synthetic complete media supplemented

with 5mM MTA and the standard amino acids and nucleotides (SC) except for the ones indicated

at the bottom of the graph. Cells were grown 48 hours and OD600 was determined.

Figure 3. Polyamine and ODC activity in wild-type and salvage pathway mutants.

A) Strains of the indicated genotype were grown in synthetic complete media to an OD of 1.0,

extracted with PCA and polyamines were measured as described in Methods. Putrescine was at

levels below detection limit (<0.5 nmoles/pg) in three of the samples. Error bars indicate

standard error of two measurements. B) ODC activity of the indicated cells were measured in

whole cell extracts as described in Methods. Error bar indicated standard error of four

measurements.

Figure 4. Steady-state ODC protein levels in salvage pathway mutants. A) ODC protein levels

were examined in total extracts made from yeast of the indicated genotype by immunoblot (see

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Materials and Methods). Yeast ODC runs at 53kD and is indicated by the arrow. The additional

bands below and slightly above ODC are presumably ODC degradation products, as they are

absent in both the wild-type and spe1 controls. B) Comparison of colony size of isogenic wild-

type and meu1∆ yeast grown on polyamine free media after three days of growth.

Figure 5. ODC activity and protein levels in response to spermidine and MTA. A) Yeast strain

AS3-2a (meu1∆), YOL052C (spe2∆), and AY32 (meu1∆ spe2∆) were grown in synthetic

complete polyamine free media in the presence or absence of 0.1 mM spermidine for 24 hours.

Extracts were prepared and analyzed for ODC activity. Error bars indicate standard error of three

measurements. B) Same strains as (A) analyzed for ODC protein levels by immunoblot. C)

Yeast strains AS3-2a (meu1∆), AS3-2a (MEU1), and YOL052C (spe2∆) were grown on

synthetic complete polyamine free media in the presence or absence of 5 mM MTA for 24 hours.

Extracts were prepared and analyzed as above.

Figure 6. Steady-state ODC protein levels in response to MTOB. Yeast strains AS3-2a

(MEU1), AS3-2a (meu1∆), YEL038W (E1∆), and YMR009W (E2∆) were grown in SC media in the

presence or absence or 0.1 mM MTOB for 24 hours. Extracts were prepared and immunoblot

analysis of ODC was preformed as described in Materials and Methods.

Figure 7. ODC activity in mammalian cells. A) MCF-7 cells were stably transfected with

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either an MTAP expressing construct (MTAP4 and MTAP8) or with a construct that expresses a

mutant non-functional MTAP (D220A4 and D220A6) (1). Cells were grown to 80%

confluency, whole cell extracts were prepared, and ODC activity was measured as described in

Methods. Error bars indicate standard error of three measurements. B) MTAP-deleted MIA

PaCa-2 pancreatic adenocarcinoma cells were plated as equal density and grown for 24 hours in

media containing the indicated levels of methionine or MTOB. Cells were then harvested,

extracts prepared, and ODC activity was measured as described in Methods. Error bars indicate

standard error of three measurements.

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Markham and Warren D. KrugerAhmad L. Subhi, Paula Diegelman, Carl W. Porter, Baiqing Tang, Zichun J. Lu, George D.

of downstream metabolitesMethylthioadenosine phosphorylase regulates ornithine decarboxylase by production

published online September 23, 2003J. Biol. Chem.

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