Anjos Jorge, Rodrigues de Sousa Helena, Roca Christophe, Cássio Fernanda, Luttik Marijke, Pronk Jack T, Salema-Oom Madalena, Gonçalves Paula
Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
Biochim Biophys Acta. 2013 Feb;1828(2):201-7. doi: 10.1016/j.bbamem.2012.08.011. Epub 2012 Aug 24.
In the model yeast Saccharomyces cerevisiae, hexose uptake is mediated exclusively by a family of facilitators (Hxt, hexose transporters). Some other Saccharomyces species (e.g. Saccharomyces bayanus and Saccharomyces pastorianus) possess, in addition, a specific fructose transporter (Fsy1, fructose symporter) that has been previously described to function as a proton symporter. In the present work, we compared growth of a yeast strain in which FSY1 occurs naturally in anaerobic, fructose- and glucose-limited chemostat cultures. Especially at low specific growth rates, fructose-proton symport was shown to have a strong impact on the biomass yield on sugar. We subsequently employed energized hybrid plasma membrane vesicles to confirm previous observations concerning the mode of operation and specificity of Fsy1 mediated transport. Surprisingly, these experiments suggested that the carrier exhibits an unusual fructose:H(+) stoichiometry of 1:2. This energetically expensive mode of operation was also found consistently in vivo, in shake flask and in chemostat cultures, and both when Fsy1 is the sole transporter and when the Hxt carriers are present. However, it is observed only when Fsy1 is operating at higher glycolytic fluxes, a situation that is normally prevented by downregulation of the gene. Taken together, our results suggest the possibility that fructose symport with more than one proton may constitute an energetically unfavorable mode of operation of the Fsy1 transporter that, in growing cultures, is prevented by transcriptional regulation.
在模式酵母酿酒酵母中，己糖摄取完全由一组转运蛋白（Hxt，己糖转运蛋白）介导。而其他一些酿酒酵母物种（如贝酵母和巴斯德酵母）还拥有一种特定的果糖转运蛋白（Fsy1，果糖同向转运蛋白），此前已证明其作为质子同向转运蛋白发挥作用。在本研究中，我们比较了FSY1天然存在的酵母菌株在厌氧、果糖和葡萄糖受限的恒化器培养中的生长情况。特别是在低比生长速率下，果糖 - 质子同向转运对糖的生物质产量有很大影响。随后，我们使用了有能量的混合质膜囊泡来证实先前关于Fsy1介导转运的操作模式和特异性的观察结果。令人惊讶的是，这些实验表明该载体呈现出1:2这种不同寻常的果糖：H(+)化学计量比。这种能量消耗大的操作模式在体内、摇瓶和恒化器培养中均一致被发现，且无论是Fsy1作为唯一转运蛋白还是同时存在Hxt载体时都是如此。然而，只有当Fsy1在较高糖酵解通量下运行时才会观察到这种情况，而这种情况通常会因基因下调而被阻止。综上所述，我们的结果表明果糖与多个质子的同向转运可能构成Fsy1转运蛋白一种能量上不利的操作模式，在生长培养中，这种情况会通过转录调控来阻止。
