Daniels Mark J, Yeager Mark
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2005 Nov 8;44(44):14443-54. doi: 10.1021/bi050565d.
The water channel protein PvTIP3;1 (alpha-TIP) is a member of the Major Intrinsic Protein membrane channel family. The in vitro activity of this aquaporin is dependent on phosphorylation, and the protein is phosphorylated in vivo by a membrane-associated Ca(2+)-dependent kinase. Mutagenesis studies have implicated three serine residues as kinase targets, but only phosphorylation of Ser7 has been observed in vivo. An atomic model of PvTIP3;1 generated by homology modeling suggested that Ser7 is the only residue that would be sterically accessible to kinases. To further explain the phosphorylation of PvTIP3;1, we overexpressed this aquaporin in the methylotrophic yeast Pichia pastoris and purified the hexahistidine-tagged protein by immobilized metal affinity chromatography. Mass spectrometry confirmed that a fraction of recombinant PvTIP3;1 was phosphorylated. Phosphatase and kinase treatments indicated that Ser7 was the only residue that could be phosphorylated. In addition, mass spectrometry indicated that the native and expressed proteins are N-terminally acetylated. This is the first demonstration that a full-length, recombinant aquaporin can be produced in yeast and authentically phosphorylated in vitro. Characterization of phosphorylation-mediated gating in PvTIP3;1 will serve as a paradigm for understanding gating mechanisms of other channels.
水通道蛋白PvTIP3;1（α-TIP）是主要内在蛋白膜通道家族的成员。这种水通道蛋白的体外活性依赖于磷酸化，并且该蛋白在体内被一种膜相关的钙依赖性激酶磷酸化。诱变研究表明有三个丝氨酸残基是激酶作用靶点，但在体内仅观察到Ser7的磷酸化。通过同源建模生成的PvTIP3;1原子模型表明，Ser7是激酶在空间上唯一可作用的残基。为了进一步解释PvTIP3;1的磷酸化，我们在甲基营养酵母毕赤酵母中过表达了这种水通道蛋白，并通过固定化金属亲和色谱法纯化了带有六组氨酸标签的蛋白。质谱分析证实，一部分重组PvTIP3;1被磷酸化。磷酸酶和激酶处理表明，Ser7是唯一可被磷酸化的残基。此外，质谱分析表明天然蛋白和表达蛋白的N端都被乙酰化。这是首次证明全长重组水通道蛋白可在酵母中产生并在体外进行真实的磷酸化。对PvTIP3;1中磷酸化介导的门控的表征将作为理解其他通道门控机制的范例。
