Echtay K S, Bienengraeber M, Klingenberg M
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, 80336 Munich, Germany.
Biochemistry. 1997 Jul 8;36(27):8253-60. doi: 10.1021/bi970513r.
For expression in Saccharomyces cerevisiae the cDNA of the uncoupling protein (UCP) of brown adipose tissue from hamster has been isolated and used to transform yeast cells. Optimized expression conditions yielded 2% of mitochondrial protein as UCP. UCP was isolated, avoiding copurification of ADP/ATP carrier and porin. Intrahelical E190, previously suggested to be the pH sensor for nucleotide binding, was neutralized to glutamine by mutagenesis. In binding titrations with [14C]guanosine 5'-triphosphate (GTP) and with fluorescent dansyl-GTP, near equal binding capacity for GTP was measured in wild-type (wt) and E190Q. The KD for GTP binding to UCP from yeast has the same strong pH dependence as the original UCP from hamster. With both [14C]GTP and dansyl-GTP, the KD in wt increased 16-19-fold from pH 6.0 to 7.5, while in E190Q this increase was only 2.5-2.9-fold. As a result, at pH 7.5, both [14C]GTP and dansyl-GTP bind 6-fold tighter to E190Q than to wt. The binding rate of GTP decreased 10-fold from pH 6.0 to 7.5 in wt and only 4-fold in E190Q. Woodward reagent K (WRK) known to interact specifically with E190 [Winkler, E., Wachter, E., and Klingenberg, M. (1997) Biochemistry 36, 148-155] abolished [14C]GTP and dansyl-GTP binding to wt UCP, whereas binding to E190Q was fully resistant to WRK. H+ and Cl- transport activity in reconstituted vesicles were the same with wt and E190Q. At pH 7.5, 5 microM GTP is unable to inhibit H+ and Cl- transport in wt but inhibits in E190Q to maximum level. The different sensitivity toward GTP versus GDP found in wt is absent in E190Q. Thus, the mutation E190Q results in the predicted gain of function in binding and proves the role of the intrahelical E190 as a pH sensor for nucleotide binding but excludes a role in transport.
为了在酿酒酵母中表达，仓鼠棕色脂肪组织解偶联蛋白（UCP）的cDNA已被分离出来，并用于转化酵母细胞。优化的表达条件下，UCP占线粒体蛋白的2%。UCP被分离出来，避免了ADP/ATP载体和孔蛋白的共纯化。先前认为是核苷酸结合pH传感器的螺旋内E190通过诱变被中和为谷氨酰胺。在用[14C]鸟苷5'-三磷酸（GTP）和荧光丹磺酰-GTP进行的结合滴定中，野生型（wt）和E190Q对GTP的结合能力几乎相等。酵母UCP与GTP结合的KD与仓鼠来源的原始UCP具有相同强烈的pH依赖性。使用[14C]GTP和丹磺酰-GTP时，wt中KD从pH 6.0到7.5增加了16-19倍，而在E190Q中这种增加仅为2.5-2.9倍。因此，在pH 7.5时，[14C]GTP和丹磺酰-GTP与E190Q的结合比与wt的结合紧密6倍。在wt中，GTP的结合速率从pH 6.0到7.5降低了10倍，而在E190Q中仅降低了4倍。已知能与E190特异性相互作用的伍德沃德试剂K（WRK）[温克勒，E.，瓦赫特，E.，和克林根贝格，M.（1997年）《生物化学》36，148-155]消除了[14C]GTP和丹磺酰-GTP与wt UCP的结合，而与E190Q的结合对WRK完全具有抗性。重组囊泡中的H+和Cl-转运活性在wt和E190Q中相同。在pH 7.5时，5 microM GTP无法抑制wt中的H+和Cl-转运，但在E190Q中可将其抑制到最大水平。E190Q中不存在wt中发现的对GTP与GDP的不同敏感性。因此，E190Q突变导致了预测的结合功能获得，并证明了螺旋内E190作为核苷酸结合pH传感器的作用，但排除了其在转运中的作用。
