His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

被引:52
作者
Lu, JMY
Bush, DR
机构
[1] Univ Illinois, Photosynth Res Unit, USDA, ARS,Madigan Labs 190, Urbana, IL 61801 USA
[2] Univ Illinois, Dept Plant Biol, Urbana, IL 61801 USA
关键词
D O I
10.1073/pnas.95.15.9025
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The proton-sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased V-max) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue, Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction.
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页码:9025 / 9030
页数:6
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