Alcohol oxidase hybrid oligomers formed in vivo and in vitro

Cell-free extract prepared from methanol-grown cells of the methylotrophic yeast Pichia methanolica showed nine multiple alcohol oxidase (AOD) bands on active staining in native polyacrylamide gel electrophoresis. Their molecular basis was investigated and two AOD-encoding genes, MOD1 and MOD2, were cloned from P. methanolica genome. When the two genes were expressed in a heterologous host, an alcohol oxidase-depleted strain of Candida boidinii(aod1Delta strain), both MOD1 and MOD2 partially complemented growth defect of the host strain on methanol. While expression of either MOD1 or MOD2 in C. boidinii aod1Delta strain gave a single AOD band corresponding to the band with the largest and smallest mobility among the nine AOD bands, respectively, co-expression of MOD1 and MOD2 resulted in multiple band formation. Mixed oligomerization of Mod1p and Mod2p in vitro also gave nine multiple bands. From these results, we concluded that the nine multiple forms of AOD observed on native-PAGE represent two homo-octamers and seven hetero-octamers of Mod1p and Mod2p. Using this zymogram analysis, we also found that Mod1p was preferably produced at low methanol concentrations in the media, while Mod2p was produced at higher methanol concentrations. This shows distinct regulatory features of the two AOD-encoding genes in this methylotrophic yeast.