Winkler K, Kienle I, Burgert M, Wagner J C, Holzer H
Biochemisches Institut, Universität Freiburg, Germany.
FEBS Lett. 1991 Oct 21;291(2):269-72. doi: 10.1016/0014-5793(91)81299-n.
We have investigated the mechanism by which heat shock conditions lead to a reversible accumulation of trehalose in growing yeast. When cells of S. cerevisiae M1 growing exponentially at 30 degrees C were shifted to 45 degrees C for 20 min, or to 39 degrees C for 40 min, the concentration of trehalose increased by about 25-fold; an effect reversed upon lowering the temperature to 30 degrees C. This was compared to the more than 50-fold rise in trehalose levels obtained upon transition from the exponential to the stationary growth phase. Whereas the latter was paralleled by a 12-fold increase in the activity of trehalose-6-phosphate synthase, no significant change in the activities of trehalose-synthesizing and -degrading enzymes was measured under heat shock conditions. Accordingly, cycloheximide did not prevent the heat-induced accumulation of trehalose. However, the concentrations of the substrates for trehalose-6-phosphate synthase, i.e. glucose-6-phosphate and UDP-glucose, were found to rise during heat shock by about 5-10-fold. Since the elevated levels of both sugars are still well below the Km-values determined for trehalose-6-phosphate synthase in vitro, they are likely to contribute to the increase in trehalose under heat shock conditions. A similar increase in the steady-state levels was obtained for other intermediates of the glycolytic pathway between glucose and triosephosphate, including ATP. This suggests that temperature-dependent changes in the kinetic parameters of glycolytic enzymes vary in steady-state levels of intermediates of sugar metabolism, including an increase of those that are required for trehalose synthesis. Trehalose, glucose-6-phosphate, UDP-glucose, and ATP, were all found to increase during the 40 min heat treatment at 39 degrees C. Since this also occurs in a mutant lacking the heat shock-induced protein HSP104 (delta hsp104), this protein cannot be involved in the accumulation of trehalose under these heat shock conditions. However, mutant delta hsp104, in contrast to the parental wild-type, was sensitive towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not the accumulation of trehalose, protects S. cerevisiae from the damage caused by a 50 degrees C treatment.
我们研究了热休克条件导致生长中的酵母中海藻糖可逆积累的机制。当在30℃下指数生长的酿酒酵母M1细胞转移至45℃20分钟或39℃40分钟时，海藻糖浓度增加约25倍；温度降至30℃时这种效应逆转。这与从指数生长期转变至稳定生长期时海藻糖水平超过50倍的升高进行了比较。后者伴随着海藻糖-6-磷酸合酶活性12倍的增加，而在热休克条件下未检测到海藻糖合成和降解酶活性的显著变化。因此，环己酰亚胺不能阻止热诱导的海藻糖积累。然而，发现热休克期间海藻糖-6-磷酸合酶的底物即6-磷酸葡萄糖和UDP-葡萄糖的浓度升高约5至10倍。由于这两种糖的升高水平仍远低于体外测定的海藻糖-6-磷酸合酶的Km值，它们可能促成了热休克条件下海藻糖的增加。对于葡萄糖和磷酸丙糖之间糖酵解途径的其他中间产物，包括ATP，稳态水平也有类似升高。这表明糖酵解酶动力学参数的温度依赖性变化会改变糖代谢中间产物的稳态水平，包括海藻糖合成所需中间产物的增加。在39℃40分钟的热处理过程中，海藻糖、6-磷酸葡萄糖、UDP-葡萄糖和ATP均被发现增加。由于在缺乏热休克诱导蛋白HSP104（Δhsp104）的突变体中也发生这种情况，该蛋白在这些热休克条件下不可能参与海藻糖的积累。然而，与亲本野生型相比，突变体Δhsp104对在50℃孵育20分钟敏感。由于该突变体也积累正常水平的海藻糖，我们得出结论，是HSP104的功能而非海藻糖的积累保护酿酒酵母免受50℃处理造成的损伤。
