Uptake of adenine by purine permeases of Coffea canephora

Purine permeases (PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives into cytosol. PUPs facilitate uptake of adenine, cytokinins and nicotine. Caffeine, a purine alkaloid derived from xanthosine, occurs in only a few eudicot species, including coffee, cacao, and tea. Although caffeine is not an endogenous metabolite in Arabidopsis and rice, AtPUP1 and OsPUP7 were suggested to transport caffeine. In this study, we identified 15 PUPs in the genome of Coffea canephora. Direct uptake measurements in yeast demonstrated that CcPUP1 and CcPUP5 facilitate adenine – but not caffeine – transport. Adenine uptake was pH-dependent, with increased activity at pH 3 and 4, and inhibited by nigericin, a potassium–proton ionophore, suggesting that CcPUP1 and CcPUP5 function as proton-symporters. Furthermore, adenine uptake was not competitively inhibited by an excess amount of caffeine, which implies that PUPs of C. canephora have evolved to become caffeine-insensitive to promote efficient uptake of adenine into cytosol. CcPUP1 & CcPUP5 distinguish adenine from caffeine. The adenine uptake activity of AtPUP1 is inhibited by caffeine.嘌呤通透酶（Purine permeases, PUPs）可介导质子偶联的核苷酸碱基及其衍生物向胞质的摄取过程。PUPs能够促进腺嘌呤、细胞分裂素与尼古丁的摄取。 咖啡因是一种源自黄苷的嘌呤生物碱，仅在咖啡、可可、茶树等少数真双子叶植物物种中存在。尽管咖啡因并非拟南芥与水稻的内源代谢物，但已有研究表明拟南芥PUP1（AtPUP1）与水稻PUP7（OsPUP7）可转运咖啡因。 本研究在小果咖啡（Coffea canephora）的基因组中鉴定出15个PUP家族基因。通过酵母直接摄取实验证实，CcPUP1与CcPUP5可介导腺嘌呤的摄取，但无法转运咖啡因。腺嘌呤的摄取具有pH依赖性，在pH 3与pH 4时活性升高，且可被钾质子离子载体尼日利亚菌素（nigericin）抑制，这表明CcPUP1与CcPUP5以质子同向转运体的方式发挥功能。 进一步研究发现，过量咖啡因并未竞争性抑制腺嘌呤的摄取，这意味着小果咖啡的PUPs已演化出对咖啡因不敏感的特性，以促进腺嘌呤向胞质的高效摄取。CcPUP1与CcPUP5可区分腺嘌呤与咖啡因。而拟南芥PUP1（AtPUP1）的腺嘌呤摄取活性可被咖啡因抑制。