Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

被引:59
作者
Baunsgaard, L
Venema, K
Axelsen, KB
Villalba, JM
Welling, A
Wollenweber, B
Palmgren, MG
机构
[1] UNIV COPENHAGEN, INST MOL BIOL, DK-1353 COPENHAGEN, DENMARK
[2] ROYAL VET & AGR UNIV, DEPT PLANT BIOL, DK-1871 FREDERIKSBERG, DENMARK
[3] UNIV CORDOBA, DEPT BIOL CELULAR, E-14004 CORDOBA, SPAIN
[4] ROYAL VET & AGR UNIV, DEPT AGR SCI, DK-1871 FREDERIKSBERG C, DENMARK
关键词
D O I
10.1046/j.1365-313X.1996.10030451.x
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Transport across the plasma membrane is driven by an electrochemical gradient of H+ ions generated by the plasma membrane proton pump (H+-ATPase). Random mutants of Arabidopsis H+-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H+-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. a Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200-500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H+ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H+ extrusion and in their ability to sustain an electrochemical H+ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H+-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H+ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plant transporters.
引用
收藏
页码:451 / 458
页数:8
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