JXB Advance Access originally published online on April 11, 2005
Journal of Experimental Botany 2005 56(416):1545-1552; doi:10.1093/jxb/eri149
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RESEARCH PAPER |
A role for phosphorylation in the regulation of the barley scutellar peptide transporter HvPTR1 by amino acids
1School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK
2Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK
* To whom correspondence should be addressed. Fax: +44 (0)161 275 3930. E-mail: mqbsswmw{at}man.ac.uk
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.
Key words: Germination, Hordeum vulgare, peptide transporter, phosphorylation, plasma membrane, scutellum
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