title: OIP30, a RuvB-like DNA helicase 2, is a potential substrate for the pollen-predominant OsCPK25/26 in rice.
abstract: Calcium ions are a well-known essential component for pollen germination and tube elongation. Several calcium-dependent protein kinases (CDPKs) are expressed predominantly in mature pollen grains and play a critical role in pollen. However, none of their interacting proteins or downstream substrates has been identified. Using yeast two-hybrid screening, we isolated OsCPK25/26-interacting protein 30 (OIP30), which is also predominantly expressed in pollen. OIP30 encodes a RuvB-like DNA helicase 2 (RuvBL2) that is well conserved in eukaryotic species from yeast to human. Yeast and Drosophila defective in RuvBL2 are non-viable. The interaction between OsCPK26 and OIP30 was confirmed by far-Western blot and pull-down experiments. OIP30 was phosphorylated in a calcium-dependent manner by OsCPK26 but not OsCPK2, which is highly similar to OsCPK26 in sequence and expression profile. OIP30 unwound partial duplex DNA with a 3' to 5' directionality by ATP hydrolysis. Concurrently, the ATPase activity of OIP30 depended on single-stranded DNA. OsCPK26 phosphorylated OIP30 and enhanced both its helicase and ATPase activity about 3-fold. OIP30 may be the potential downstream substrate for OsCPK25/26 in pollen. This report characterizes a RuvBL in plants and links its activities with its upstream regulator.
authors.affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, 40227 ROC.
authors.forename: Cheng-Wei
authors.initials: CW
authors.lastname: Wang
authors.forename: Wan-Chieh
authors.initials: WC
authors.lastname: Chen
authors.forename: Li-Jing
authors.initials: LJ
authors.lastname: Lin
authors.forename: Chung-Tsai
authors.initials: CT
authors.lastname: Lee
authors.forename: Tung-Hai
authors.initials: TH
authors.lastname: Tseng
authors.forename: Wei-Ming
authors.initials: WM
authors.lastname: Leu
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D026481:Blotting, Far-Western; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011058:Pollen / Q000201:enzymology* / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D020798:Two-Hybrid System Techniques
chemical_list: D004277:DNA, Single-Stranded; D010940:Plant Proteins; D018749:RNA, Plant; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D002118:Calcium