Pahlman A K, Granath K, Ansell R, Hohmann S, Adler L
Department of Cell and Molecular Biology, Microbiology, Göteborg University, Box 463, SE 40530 Göteborg, Sweden.
J Biol Chem. 2001 Feb 2;276(5):3555-63. doi: 10.1074/jbc.M007164200. Epub 2000 Oct 31.
We have characterized the strongly homologous GPP1/RHR2 and GPP2/HOR2 genes, encoding isoforms of glycerol 3-phosphatase. Mutants lacking both GPP1 and GPP2 are devoid of glycerol 3-phosphatase activity and produce only a small amount of glycerol, confirming the essential role for this enzyme in glycerol biosynthesis. Overproduction of Gpp1p and Gpp2p did not significantly enhance glycerol production, indicating that glycerol phosphatase is not rate-limiting for glycerol production. Previous studies have shown that expression of both GPP1 and GPP2 is induced under hyperosmotic stress and that induction partially depends on the HOG (high osmolarity glycerol) pathway. We here show that expression of GPP1 is strongly decreased in strains having low protein kinase A activity, although it is still responsive to osmotic stress. The gpp1Delta/gpp2Delta double mutant is hypersensitive to high osmolarity, whereas the single mutants remain unaffected, indicating GPP1 and GPP2 substitute well for each other. Transfer to anaerobic conditions does not affect expression of GPP2, whereas GPP1 is transiently induced, and mutants lacking GPP1 show poor anaerobic growth. All gpp mutants show increased levels of glycerol 3-phosphate, which is especially pronounced when gpp1Delta and gpp1Delta/gpp2Delta mutants are transferred to anaerobic conditions. The addition of acetaldehyde, a strong oxidizer of NADH, leads to decreased glycerol 3-phosphate levels and restored anaerobic growth of the gpp1Delta/gpp2Delta mutant, indicating that the anaerobic accumulation of NADH causes glycerol 3-phosphate to reach growth-inhibiting levels. We also found the gpp1Delta/gpp2Delta mutant is hypersensitive to the superoxide anion generator, paraquat. Consistent with a role for glycerol 3-phosphatase in protection against oxidative stress, expression of GPP2 is induced in the presence of paraquat. This induction was only marginally affected by the general stress-response transcriptional factors Msn2p/4p or protein kinase A activity. We conclude that glycerol metabolism plays multiple roles in yeast adaptation to altered growth conditions, explaining the complex regulation of glycerol biosynthesis genes.
我们已经对编码甘油 3 - 磷酸酶同工型的高度同源的 GPP1/RHR2 和 GPP2/HOR2 基因进行了表征。缺乏 GPP1 和 GPP2 的突变体没有甘油 3 - 磷酸酶活性，并且仅产生少量甘油，这证实了该酶在甘油生物合成中的重要作用。Gpp1p 和 Gpp2p 的过量表达并未显著提高甘油产量，表明甘油磷酸酶不是甘油生产的限速因素。先前的研究表明，GPP1 和 GPP2 的表达在高渗胁迫下均被诱导，并且这种诱导部分依赖于 HOG（高渗甘油）途径。我们在此表明，在蛋白激酶 A 活性较低的菌株中，GPP1 的表达显著降低，尽管它仍然对渗透胁迫有反应。gpp1Delta/gpp2Delta 双突变体对高渗敏感，而单突变体不受影响，表明 GPP1 和 GPP2 可以很好地相互替代。转移到厌氧条件下不影响 GPP2 的表达，而 GPP1 被短暂诱导，并且缺乏 GPP1 的突变体显示出较差的厌氧生长。所有 gpp 突变体中甘油 3 - 磷酸的水平都有所增加，当 gpp1Delta 和 gpp1Delta/gpp2Delta 突变体转移到厌氧条件下时尤其明显。添加乙醛（一种 NADH 的强氧化剂）会导致甘油 3 - 磷酸水平降低，并恢复 gpp1Delta/gpp2Delta 突变体的厌氧生长，这表明 NADH 的厌氧积累会使甘油 3 - 磷酸达到抑制生长的水平。我们还发现 gpp1Delta/gpp2Delta 突变体对超氧化物阴离子产生剂百草枯敏感。与甘油 3 - 磷酸酶在抵抗氧化应激中的作用一致，在百草枯存在的情况下，GPP2 的表达被诱导。这种诱导仅受到一般应激反应转录因子 Msn2p/4p 或蛋白激酶 A 活性的轻微影响。我们得出结论，甘油代谢在酵母适应生长条件变化中起多种作用，这解释了甘油生物合成基因的复杂调控。
