Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin.
PV72, a type I membrane protein with three epidermal-growth factor (EGF)-like motifs, was found to be localized on the membranes of the precursor-accumulating (PAC) vesicles that accumulated precursors of various seed storage proteins. To clarify the function of PV72 as a sorting receptor, we expressed four modified PV72s and analyzed their ability to bind the internal propeptide (the 2S-I peptide) of pro2S albumin by affinity chromatography and surface plasmon resonance. The recombinant PV72 specifically bound to the 2S-I peptide with a K(D) value of 0.2 microm, which was low enough for it to function as a receptor. The EGF-like motifs modulated the Ca(2+)-dependent conformational change of PV72 to form a functional pocket for the ligand binding. The binding of Ca(2+) stabilizes the receptor-ligand complex even at pH 4.0. The association and dissociation of PV72 with the ligand is modulated by the Ca(2+) concentration (EC(50) value = 40 microm) rather than the environmental pH. Overall results suggest that Ca(2+) regulates the vacuolar sorting mechanism in higher plants.
Watanabe
Etsuko
E
Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
Shimada
Tomoo
T
Kuroyanagi
Miwa
M
Nishimura
Mikio
M
Hara-Nishimura
Ikuko
I
D055516:2S Albumins, Plant; D020816:Amino Acid Motifs; D052179:Antigens, Plant; D002118:Calcium / Q000378:metabolism*; D028464:Cucurbita / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007700:Kinetics; D008024:Ligands; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D055516:2S Albumins, Plant; D052179:Antigens, Plant; D006023:Glycoproteins; D008024:Ligands; C111597:PAC vesicle 72-kDa protein, Cucurbita; D010455:Peptides; D010940:Plant Proteins; D011498:Protein Precursors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D002118:Calcium