The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export
The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export
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DOI：
10.1074/jbc.m100810200
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发表时间：
2001-08-03
影响因子：
4.8
通讯作者：
Maloney, PC
中科院分区：
文献类型：
--
作者：
Ketchum, CJ;Schmidt, WK;Maloney, PC
ATP结合盒（ABC）蛋白转运多种底物。据推测，这些蛋白质偶联ATP水解底物运输，但ATP酶活性已被证明只有少数。为了提供直接的证据，这种活动在步骤6p，酵母ABC蛋白质所需的出口的a-因子交配信息素，我们建立了条件，纯化步骤6p的生化量从酵母和Sf 9昆虫细胞。基础ATP酶活性的纯化和重建的Ste 6p（Vmax = 18 nmol/ mg/min; K-m为MgATP = 0.2 mm）相比，有利地与其他几个ABC蛋白质，并抑制原钒酸盐在诊断的ABC转运蛋白（表观K-I = 12 μ m）的配置文件。在添加合成或天然形式的α-因子后观察到适度的刺激（类似于40%）。我们还使用S-叠氮基[α- P-32]ATP结合和钒酸盐捕获测定来检查野生型Ste 6p和两种不同的双突变体（G392 V/G1087 V和G509 D/G11931）的行为，所述两种不同的双突变体先前显示为在体内交配缺陷。这两种突变体显示出减弱的能力，水解ATP，与后者解偶联从信息素运输。我们的结论是，STE 6P催化ATP水解耦合到a-因子运输，这反过来又促进交配。
ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V-max = 18 nmol/ mg/min; K-m for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K-I = 12 mum). Modest stimulation (similar to 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an S-azido[alpha- P-32]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G11931)) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.