The barley scutellar peptide transporter: biochemical characterization andlocalization to the plasma membrane

Thiol-affinity labelling was used to identify and characterize components o f the peptide transport system in the barley (Hordeum vulgare) scutellar ep ithelium. SDS-PAGE and 2D-PAGE in conjunction with fluorography were used t o study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were i dentified using a strategy devized to label substrate protectable protein w ith the thiol specific reagent [C-14] N-ethylmaleimide (NEM), The scutellar plasma membrane is the anticipated site of transporters involved in the mo bilization of endosperm storage reserves in the germinating barley grain. T he subcellular localization of these proteins to the plasma membrane was de monstrated by thiol-affinity labelling of high purity plasma membrane vesic les isolated from barley scutellar tissue, A peptide transporter, HvPTR1, s pecific to the barley scutellum has recently been cloned in this laboratory . A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, w as identified by [C-14]NEM labelling studies of Xenopus laevis oocytes expr essing HvPTR1 cRNA, but not water injected controls. Peptide antiserum rais ed to HvPTR1 also cross-reacted with a 66 kDa membrane protein in barley sc utellar tissue, This confirms that the 66 kDa protein identified here by th iol-affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membr ane of scutellar epithelial cells during germination.