Smits H P, Smits G J, Postma P W, Walsh M C, van Dam K
E. C. Slater Institute, BioCentrum Amsterdam, University of Amsterdam, The Netherlands.
Yeast. 1996 Apr;12(5):439-47. doi: 10.1002/(SICI)1097-0061(199604)12:5%3C439::AID-YEA925%3E3.0.CO;2-W.
Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.
酿酒酵母中的葡萄糖摄取被认为由两个动力学上可区分的组分组成，其亲和力在以葡萄糖为碳源生长期间受到调节。据报道，三己糖激酶缺失突变体不表现出高亲和力的葡萄糖摄取。这就提出了一个问题，即高亲和力的葡萄糖摄取是否以及如何与葡萄糖磷酸化酶的存在相关。在本研究中，使用快速摄取方法测定了在甘油或半乳糖上生长的野生型细胞和己糖激酶缺失突变体细胞中葡萄糖摄取的动力学。在野生型细胞中，在5秒或200毫秒内测得的葡萄糖摄取表现出高亲和力。相比之下，在己糖激酶缺失突变体细胞中，葡萄糖摄取的表观亲和力取决于测量摄取的时间尺度。在5秒尺度上的测量显示表观低亲和力摄取，而在200毫秒尺度上的测量显示高亲和力摄取。后者的亲和力和最大速率与野生型细胞中的相当。使用对称转运体的简单模型，可以模拟实验确定的表观亲和力与所用时间尺度之间的关系。结果表明，高亲和力葡萄糖转运不一定依赖于葡萄糖磷酸化酶的存在。表观低亲和力摄取动力学可能是由于磷酸化作用去除细胞内游离葡萄糖的速率不足所致。本研究强调了区分转运体和代谢对酵母中糖摄取表观动力学影响的必要性。
