43[Noda Institute for Scientific Research GRANT]2006 Young Investigator Research Grant
Outline of Research Result

Analysis of the Effect of S-adenosylmethionine on Cell
Growth in Yeast
Masaki MIZUNUMA
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University

Research aims
S-adenosyl-L-methionine (AdoMet) is a high-energy sulfonium compound used in most of the numerous biologicalmethylation reactions. An alteration in the availability ofAdoMet has a profound effect on cell growth. In a previousstudy, we isolated a mutant allele of Saccharomyces cerevisiae gene SAH1 encoding S-adenosyl-L-homocysteine(AdoHcy) hydrolase, as a mutation that suppressed theCa2-sensitive phenotypes of the zds1D strain, such as theCa2-induced, G2 cell-cycle arrest and polarized bud
growth1,2). Interestingly, the mutation (sah1-1) led the cells
to accumulate AdoMet besides AdoHcy, the substrate ofthis hydrolase. A case of AdoHcy hydrolase deficiency inhumans was also reported to cause the patient’s leukocyte
DNA to be hyper-methylated relative to the control level
due to extreme levels of AdoHcy3). Similar to that in yeast,the defect in AdoHcy hydrolase in humans causes simultaneous elevation of AdoHcy and AdoMet. Thus the sah1-1
mutant is a useful model to deepen our knowledge of theconsequences of cellular accumulation of AdoHcy andAdoMet. The aim of this study was to investigate the phenotypes of the sah1-1 mutant and elucidate the role ofAdoMet and AdoHcy in yeast cell growth.
Methods
Saccharomyces cerevisiae (wild-type, sah1-1 or vps33D )cells were grown in rich medium (YPD). Unless otherwise
indicated, all yeast strains were grown routinely at 25°C.
Extraction of AdoMet and AdoHcy was carried out as described previously1). Determination of AdoMet and AdoHcy was performed by capillary electrophoresis using aWaters Capillary Ion Analyzer (Milford, MA, USA) withan Accusep fused silica gel column (60-cm total length and75-m m ID)1). RNA isolation and DNA microarray profilingexperiments were performed as described earlier4).
Results
In previous work, we showed that the cellular content ofAdoHcy and AdoMet in the sah1-1 strain was elevated in
YPD medium at 25°C, a permissive temperature for sah1mutants1). It was earlier shown that hyper-accumulation of
AdoMet caused a growth defect in a strain in which theVPS33 gene encoding a non-essential Class C vacuolar
sorting protein had been deleted. Therefore, we constructeda sah1-1 vps33D strain, and examined its growth. As expected, the sah1-1 vps33D strain was not viable, suggestingthat intracellular AdoHcy and AdoMet had accumulated invacuoles and that high levels of AdoHcy and AdoMet were
harmful to yeast cell growth.
We have shown that the sah1-1 mutant exhibited a slow
growth phenotype at all temperatures examined (1437°C), with more severe retardation of growth at extremetemperatures. We expected that the intracellular content ofAdoHcy and AdoMet in the sah1-1 strain would be elevated at a high temperature. Therefore we determined thecellular content of AdoHcy and AdoMet in the sah1-1
strain in YPD medium at 37°C, a non-permissive temperature for sah1 mutants. The amounts of AdoHcy andAdoMet in the cell extracts were determined by capillaryelectrophoresis. The cellular contents of AdoHcy andAdoMet in the sah1-1 strain in YPD medium were about
2.5- and 6-fold, respectively, higher at 37°C than at 25°C.
In the sah1-1 strain, the cellular accumulation of the AdoHcy was accompanied by the remarkable accumulation ofAdoMet. It is known that the growth of a cystathionine b synthase mutant becomes inhibited when the intracellular
AdoMet/AdoHcy ratio drops below 1.55). We previously
showed that the AdoMet/AdoHcy ratio in the sah1-1 cells
grown in YPD medium at 25°C was 6.31). At 37°C, thisratio was 13.4. The elevation of the AdoMet/AdoHcy ratioat the high temperature in sah1-1 cells would seem to be
the result of up-regulation of the AdoMet production frommethionine by some unknown mechanism to cope with thecritical conditions due to the detrimental accumulation ofAdoHcy. Further, we found that unbudded enlarged G1 cells
accumulated upon shifting of the sah1-1 strain to 37°C. To
validate the above result, we assayed the distribution of thecells throughout the cell cycle via fluorescence-activatedcell sorting (FACS), measuring the numbers of cells with 1(1C) and 2 (2C) copies of the chromosomes. The FACS
data confirmed that sah1-1 cells in the YPD medium at37°C were delayed in the G1 phase.
To test if elevation of the AdoHcy and AdoMet contentsof the sah1-1 cells affected gene transcription, we conducted DNA microarray analysis. Inspection of the full data
set revealed several characteristics of the sah1-1-dependent
gene. We identified genes whose expression was increased

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more than 2-fold in the sah1-1 cells at 25°C. In total, 64
genes were induced more than 2-fold; and they were those
involved in lipid biosynthesis, phosphate/polyphosphatebiosynthesis, and methionine biosynthesis. Interestingly,
SAH1 was also up-regulated at the transcriptional level.
Next, we carried out the DNA microarray analysis on thesah1-1 cells at 37°C. Unfortunately, we could not detect
any signal for some unknown reason.
Conclusion
The sah1-1 mutation, an allele of the SAH1 gene, wascharacterized in this study. In the sah1-1 strain, the cellularaccumulation of the AdoHcy, the substrate of AdoHcy hydrolase, was accompanied by the remarkable accumulationof AdoMet. This phenotype was enhanced by a shift to ahigher temperature (37°C). At the higher temperature, thesah1-1 cells were delayed in the G1 phase of the cell cycle.
DNA microarray analysis demonstrated that accumulationof AdoHcy and AdoMet in the sah1-1 cells was due to theup-regulation of the mRNA of enzymes involved in methionine biosynthesis. Further analyses are necessary to expand
our knowledge of the consequences of the cellular accumu-lation of AdoHcy and AdoMet.
References
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