Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.
Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.
Azad
Abul Kalam
AK
Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu, Shimane 690-8504, Japan.
Sawa
Yoshihiro
Y
Ishikawa
Takahiro
T
Shibata
Hitoshi
H
D020346:Aquaporins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D009997:Osmotic Pressure; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013104:Spheroplasts / Q000235:genetics; D031447:Tulipa / Q000235:genetics*
D020346:Aquaporins; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases