Phosphorylation of aquaporin PvTIP3;1 defined by mass spectrometry and molecular modeling.
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.
Daniels
Mark J
MJ
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Yeager
Mark
M
D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism*
D020346:Aquaporins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; C078044:alpha-TIP protein, Phaseolus vulgaris; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases