A role for phosphorylation in the regulation of the barley scutellar peptide transporter HvPTR1 by amino acids.
Protein reserves in the cereal endosperm are sequentially degraded to small peptides and amino acids during germination and these are translocated across the scutellum to support growth of the embryo. Peptide transport in the germinating barley grain is mediated by specific carriers localized to the plasma membrane of the scutellar epithelium. In isolated barley embryos peptide transport is rapidly inhibited by amino acid concentrations comparable with those found in the post-germination barley grain. However, this inhibition of HvPTR1 activity is not effected at either the transcriptional or translational level. The protein phosphatase inhibitor okadaic acid repressed transport of Ala-[14C]Phe, but not [14C]Ala, into the barley scutellar epithelium. In vivo [32P]orthophosphate labelling studies of barley scutellar tissue in combination with immunoprecipitation studies using antiserum raised to HvPTR1 showed that HvPTR1 (66 kDa) is phosphorylated in the presence of amino acids. Immunopurified HvPTR1 was further demonstrated to be phosphorylated on serine residues. Digestion with the N-glycosidase enzyme PNGase F results in a shift in the molecular mass of the protein by 10 kDa, indicating that HvPTR1 is an N-linked glycoprotein. These results provide strong circumstantial evidence that HvPTR1 peptide transport activity in the germinating barley grain is regulated at the post-translational level by phosphorylation in response to rising levels of amino acids emanating from the endosperm as a result of storage protein breakdown and mobilization. This is potentially an important element in balancing the flux of organic nitrogen and carbon from the endosperm to embryo during germination and seedling establishment.
Waterworth
Wanda M
WM
School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. mqbsswmw@man.ac.uk
Ashley
Merewyn K
MK
West
Christopher E
CE
Sunderland
Paul A
PA
Bray
Clifford M
CM
D000596:Amino Acids / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D001693:Biological Transport, Active / Q000502:physiology; D018506:Gene Expression Regulation, Plant; D018525:Germination / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology* / Q000378:metabolism; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013997:Time Factors
D000596:Amino Acids; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D010455:Peptides; D010940:Plant Proteins; C114566:Ptr1 protein, Hordeum vulgare; D005947:Glucose