Developmental expression of three mungbean Hsc70s and substrate-binding specificity of the encoded proteins.
We isolated three mungbean Hsc70 cDNAs (VrHsc70-1, 70-2 and 70-3) and characterized their developmental expression at both the transcript and protein levels. We also characterized the binding specificity between each VrHsc70 protein and its potential substrates. RNase protection assays showed that these three cytosolic VrHsc70 genes were expressed similarly in all organs at all times during the mungbean life cycle, except at the initiation of germination and during late seed embryogenesis. Western blotting analyses showed that a different group of cytosolic VrHsc70 proteins accumulated in dehydrated seeds during seed maturation and the accumulated proteins remained high during the early stages of germination. Binding specificities of these three mungbean Hsc70s were determined using the C-terminal 30 kDa of the three VrHsc70s to select bound heptapeptides using phage display screening, and were confirmed by ELISA. We found that the heptapeptides, KVWVLPI, KLWVIPQ and YAPLSRL, specifically bound to the C-terminal 30 kDa region of VrHsc70-1, 70-2 and 70-3, respectively. The hydrophobic residues in the core of the heptapeptides, as well as residues 6 and 7, might contribute to the binding specificity. Our results indicate that the function of these three VrHsc70s may not be important in seed maturation or in desiccation tolerance, but are more likely involved in normal growth and development.
Chen
Yi-Jiun
YJ
Department of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, R.O.C.
Wu
Miin-Feng
MF
Yu
Yueh-hsiang
YH
Tam
Ming F
MF
Lin
Tsai-Yun
TY
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D007887:Fabaceae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D018525:Germination; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins