Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement.
In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.
Taoka
Ken-Ichiro
K
Section of Plant Biology, College of Biological Sciences, University of California, Davis, CA 95616, USA.
Ham
Byung-Kook
BK
Xoconostle-Cázares
Beatriz
B
Rojas
Maria R
MR
Lucas
William J
WJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001692:Biological Transport; D028464:Cucurbita / Q000166:cytology* / Q000378:metabolism*; D006031:Glycosylation; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D052585:Phloem / Q000166:cytology* / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D031425:Plasmodesmata / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D012694:Serine