Single replacement constructs of all hydroxyl, basic, and acidic amino acids identify new function and structure-sensitive regions of the mitochondrial phosphate transport protein.
Single replacement constructs of all hydroxyl, basic, and acidic amino acids identify new function and structure-sensitive regions of the mitochondrial phosphate transport protein.
复制标题
所有羟基、碱性和酸性氨基酸的单一替换构建体鉴定了线粒体磷酸转运蛋白的新功能和结构敏感区域。
DOI：
10.1021/bi0117551
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Haefele,Amanda
中科院分区：
文献类型：
--
作者：
Wohlrab,Hartmut;Annese,Vincent;Haefele,Amanda
磷酸盐转运蛋白(PTP)催化磷酸盐的质子共转运进入线粒体基质。它的功能是同源二聚体，因此磷酸盐和质子孔的残留物在初级序列中有些分散。每个PTP亚基有71个新的单一突变，其所有的羟基、碱性和酸性残基现在都已被取代，以识别这些必需残基。我们测定了这些突变体的pH梯度依赖的单向磷酸盐转运活性的初始速率和脂质体掺入效率(LIE)。Thr79、Tyr83、Lys90、Tyr94和Lys98的单一突变使转运失活。这些残基之间的间距意味着它们位于跨膜(TM)螺旋B的同一面，需要将其目前的模型C-末端结构域延长10个残基。这一延伸很好地叠加在较短的牛PTP螺旋B上，留下了酵母螺旋B N末端的15个残基的疏水延伸。这与酵母PTP的螺旋D和F区相似。只有一个转运抑制突变位于环内： Ser158Thr位于螺旋C和D之间的矩阵环中。所有其他转运抑制突变都位于TM螺旋内。产生-lt；6%的突变都在螺旋内，只有四个除外。四个例外是靠近PTP N-末端的Tyr12Ala和螺旋C和D之间的环中的Arg159Ala、Glu163Gln和Glu164Gln。在螺旋E的N-末端，超过Thr214的PTP C-末端片段有11个突变，Lies>；20%，没有Lie<；6%。20%的突变位于除TM螺旋D外的所有TM螺旋的末端附近，只有少数突变改变了PTP结构(LiE)，也影响了PTP的转运活性。一个新的观察结果是，Ser4Ala阻止了PTP细菌包涵体的形成。
The phosphate transport protein (PTP) catalyzes the proton cotransport of phosphate into the mitochondrial matrix. It functions as a homodimer, and thus residues of the phosphate and proton pores are somewhat scattered throughout the primary sequence. With 71 new single mutation per subunit PTPs, all its hydroxyl, basic, and acidic residues have now been replaced to identify these essential residues. We assayed the initial rate of pH gradient-dependent unidirectional phosphate transport activity and the liposome incorporation efficiency (LIE) of these mutants. Single mutations of Thr79, Tyr83, Lys90, Tyr94, and Lys98 inactivate transport. The spacings between these residues imply that they are located along the same face of transmembrane (TM) helix B, requiring an extension of its current model C-terminal domain by 10 residues. This extension superposes very well onto the shorter bovine PTP helix B, leaving a 15-residue hydrophobic extension of the yeast helix B N-terminus. This is similar to the helix D and F regions of the yeast PTP. Only one transport-inhibiting mutation is located within loops: Ser158Thr in the matrix loop between helices C and D. All other transport-inhibiting mutations are located within the TM helices. Mutations that yield LIEs of <6% are all, except for four, within helices. The four exceptions are Tyr12Ala near the PTP N-terminus and Arg159Ala, Glu163Gln, and Glu164Gln in the loop between helices C and D. The PTP C-terminal segment beyond Thr214 at the N-terminus of helix E has 11 mutations with LIEs >20% and none with LIE <6%. Mutations with LIEs >20% are located near the ends of all the TM helices except TM helix D. Only a few mutations alter PTP structure (LIE) and also affect PTP transport activity. A novel observation is that Ser4Ala blocks the formation of PTP bacterial inclusion bodies.