Phosphorylation of Aquaporin PvTIP3;l Defined by Mass Spectrometry and Molecular Modeling

摘要

The water channel protein PvTIP3;l (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;l 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;l,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;l 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;l will serve as a paradigm for understanding gating mechanisms of other channels.