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NISR RESEARCH GRANT
2002 Young Investigator Research Grant

Study on Oxygen Sensing in Yeast
Tomoyuki NAKAGAWA
Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture
Most organisms have respiratory system for energy generation, and use oxygen as electron acceptor for respiration.
However, on being shifted from an aerobic to an oxygendepleted environment, many processes within the cell haveto adapt to the new conditions. For example, in the yeast
Saccharomyces cerevisiae, energy generation exchanges
fermentation from respiratory on an anaerobic condition.
Moreover, the place, where oxygen-requiring reactions
take place, is not only the respiratory chain, but also otherorganelle, e.g.; peroxisome, on which b -oxidation and otheroxidative pathways are located. Therefore, organisms must
recognize oxygen concentrations on growth condition, andmust maintain the active balance between respiration andother metabolism using oxygen, making adaptation to theconcomitantly changing environmental conditions possible.
On the other hand, the methylotrophic yeast, which isable to utilize methanol as a sole carbon source, proliferated large peroxisomes, in which alcohol oxidase (AOD),
key enzyme on methanol metabolism, is located. In
methanol metabolism, oxygen is consumed by AOD on peroxisome, as well as by respiratory on mitochondrion.
Therefore, it seems that methanol-metabolic pathway isable to use as model pathway for examining the molecularmechanism of oxygen sensing. In this study, we investigated regulation of AOD-gene expression by oxygen inmethylotrophic yeast Pichia methanolica.
1.

Expresstion of AOD isozyme by oxygen

We had reported that P. methanolica has nine AODisozyme, which is octamer consisted of two subunits encoded by two AOD genes, MOD1 and MOD2, and the bandprofile of an AOD zymogram reflects the composition ratioof two subunit species (1). At first, we observed effect ofoxygen concentrations on induction of AOD isozyme. The
AOD isozyme was not induced under anaerobic conditions,although showed nine forms on aerobic condition. Moreover, the composition of the AOD-isozyme species responded to the oxygen concentration. With an increase in
the oxygen concentration, the ratio of Mod2p to Mod1p increased (Fig. 1A). These zymogram patterns and the geneexpression patterns of MOD1 and MOD2 coincided eachother. With an increase in the oxygen concentration, theMOD1 mRNA level decreased and the level of MOD2 expression was becoming dominant. Therefore, the regulationof AOD isozyme by oxygen was confirmed to be controlled
mainly at the mRNA level.
2. Regulation of function balance between AOD
isozyme and respiratory
It seems that the balance of oxygen consumption between AOD activity and respiratory function is importantfor methanol metabolism. Next, we added the respiratory
inhibitor, i.e., antimycin A and sodium azide, to AOD-inducible condition, in order to observe effect of respiratory
function on induction of AOD isozyme. Presence of respiratory inhibitors repressed induction of AOD isozyme.
With an increase in inhibitor concentration the level ofMod1p was becoming dominant, although Mod2p wasdominant on low concentration of respiratory inhibitor.
Moreover, zymogram pattern by addition of inhibitor isvery similar to that showed by the change of oxygen concentrations (Fig. 1B). These results indicate that respiratory

Fig. 1. Zymogram patterns of AOD-isozyme complexes from the P. methanolica induced with (A) various concentrations of oxygen, and (B) various concentrations of sodium azide. Electrophoresis was carried out on a 5% polyacrylamide gel. The oxygen concentrations were controlled by shaking speed: lane
1, anaerobic condition; lane 2, 50 rpm; lane 3, 100 rpm; and lane 4, 150 rpm. The sodium azide concentrations were; lane 5, 0%; lane 6, 1103%; lane 7,2103%; and lane 8, 3103%.

89function is essential for the induction of AOD isozyme.
3. Oxygen-metabolic balance and crosstalk between
organelles
In this study, we showed that oxygen sensing in AODgene expression involves respiratory function, and it seems
that mitochondrion releases some signals that function in
the AOD-gene expression. That is to say, AOD-gene ex-pression is controlled according as respiratory function, andthere are crosstalks for oxygen sensing between some organelles, e.g.; mitochondrion-nuclear-peroxisome, to maintain oxygen-metabolic balance in the cell.
1) Nakagawa T, Mukaiyama H, Yurimoto H, Sakai Y, Kato N. 1999.
Alcohol oxidase hybrid oligomers formed in vivo and in vitro. Yeast
15: 1223–1230.