Esther Charles R, Sesma Juliana I, Dohlman Henrik G, Ault Addison D, Clas Marién L, Lazarowski Eduardo R, Boucher Richard C
Division of Pediatric Pulmonology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Biochemistry. 2008 Sep 2;47(35):9269-78. doi: 10.1021/bi800855k. Epub 2008 Aug 12.
Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y 14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Delta was reduced by deletion of the YEA4 UDP- N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation.
细胞外的UDP-葡萄糖是一种天然的嘌呤能受体激动剂，但其细胞释放机制尚不清楚。我们在酿酒酵母中研究了这些机制，酿酒酵母是一种能释放另一种嘌呤能激动剂ATP的简单模式生物。与ATP类似，酿酒酵母释放UDP-葡萄糖的速率随时间呈线性变化。然而，与ATP释放不同的是，UDP-葡萄糖的释放不依赖于葡萄糖刺激。通过证明ATP释放中明显的葡萄糖刺激反映了腺嘌呤核苷酸细胞内模式的葡萄糖依赖性变化，解决了这一差异，在没有葡萄糖的情况下，AMP释放占主导地位。事实上，总的腺嘌呤核苷酸释放，与UDP-葡萄糖释放一样，在短期内不会随葡萄糖浓度而变化。通过对缺失突变体的分析，借助基于异源表达的人P2Y14受体信号传导开发的一种新的UDP-葡萄糖生物测定法，探索了UDP-葡萄糖释放的遗传基础。使用这种测定法，在缺乏假定的高尔基体核苷酸糖转运蛋白YMD8的突变体中，UDP-葡萄糖释放速率升高。通过缺失YEA4 UDP-N-乙酰葡糖胺或HUT1 UDP-半乳糖转运蛋白，可降低ymd8Delta中UDP-葡萄糖释放速率的增加，而YEA4或HUT1的过表达则增加了UDP-葡萄糖释放速率。这些发现表明存在一种类似于ATP的胞吐释放机制，这一结论得到了分泌抑制剂布雷菲德菌素A作用下ATP、AMP和UDP-葡萄糖释放速率降低的支持。这些研究证明了分泌途径参与酵母中核苷酸和核苷酸糖的外排，并为进一步研究提供了一个强大的模型系统。
