Biochemical studies on PV72, a vacuolar sorting receptor of higher plants

In higher plants, there are two types of vacuoles: protein storage vacuoles, which accumulate seed storage proteins, and lytic vacuoles, which are analogous to lysosomes and vacuoles in mammals and yeast. Vacuolar proteins are synthesized at rough endoplasmic reticulum (rER) and transported to respective vacuoles. Unlike the transport of lytic proteins, the molecular mechanism of seed storage proteins is still unclear. Precursor accumulating (PAC) vesicles that had accumulated large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seed. PV72, a type I membrane protein with three epidermal- growth factor (EGF)-like motifs, was found to be localized on the membranes of the PAC vesicles that accumulated precursors of various seed storage proteins. PV72 is composed of lumenal domain, transmembrane domain, and cytiplasmic domain. The luminal domain contains three EGF-like motifs. The third EGF-like motif was a Ca2+ -binding type. Vacuolar sorting receptors for seed storage proteins have not been found. To clarify the function of PV72 as a vacuolar sorting receptor for seed storage protein, they performed molecular studies on PV72. They found that the lumenal domain of PV72 (rPV72) expressed in insect cells bound to proprotein precursor of 2S albumin in a Ca2+-dependent manner. They analyzed the domain of PV72 by expressing modified PV72s, rPV72Δ3, rPV72Δ2,3, and rPV72Δ1,2,3 in insect cells. Then they examined, these modified PV72s with the internal propeptide (the 2S-I peptide) of pro2S albumin, a seed storage protein precursor, by affinity chromatography and surface plasmon resonance analysis. rPV72 specifically bound to the 2S-I peptide with a KD value of 0.2 μM, which was low enough for it to function as a receptor. Lytic type sorting receptors have been reported to bind to ligand in a pH-dependent manner. Thus they examined the possibility of pH as a regulation factor of interaction and clarified that the binding of Ca2+ stabilizes the receptor-ligand complex even at pH 4.0. They focused on Ca2+ as the regulatory factor for the association and dissociation of PV72 with the ligand and clarified that PV72 bound to the ligand in a Ca2+-dependent manner. Furthermore, they clarified that the EGF-like motifs modulated the Ca2+-dependent conformational change of PV72 to form a functional pocket for the ligand binding. The association and dissociation of PV72 with the ligand is modulated by the Ca2+ concentration (EC50 value = 40 μM) rather than the environmental pH. The overall results suggested that PV72 functions as a sorting receptor for pro2S albumin and that Ca2+ regulates the sorting mechanism. As described above, PV72 seemed to be a key molecule for intracellular transport of vacuolar proteins. Thus, they further characterized authentic PV72 and PV82 derived from pumpkin and found that PV72 and PV82 showed different characters. Further, they analyzed the binding of rAtELP that is reported as the sorting receptor for lytic protease. They found that rAtELP bound to the AtPAP peptide in a Ca2+-dependent manner rather than pH. They demonstrated that Ca2+ is a key regulator for transport of vacuolar proteins in higher plants. The natural ligand of each receptor is not well known. BP-80 that had been reported as lytic-type sorting receptor bound both to the peptide derived from aleurain and to that from Brazil nuts 2Salbumin. PV72 which functions as storage protein type sorting receptor also bound both to the peptide derived from 2S albumin and to the peptide derived from aleurain. These results indicated these receptors had similar binding site and bound to various types of ligands including lytic protease and storage protein. In contrast to well characterization in vitro, the demonstration for the in vivo function of vacuolar sorting receptors has been poorly done. Then, I performed the in vivo demonstration of PV72 in Arabidopsis cells. However, the demonstration of the sorting to vacuoles by such receptor molecules has not been done in plant cells. To elucidate a physiological function of such putative vacuolar sorting receptor in plant cells, they produced transgenic Arabidopsis plants that expressed a fusion protein (PV72-HDEL) composed of the lumen domain of PV72 with a His-tag and an endoplasmic reticulum (ER)-retention signal, HDEL. The ectopic overexpression of PV72-HDEL induced the accumulation of a precursor of a cysteine proteinase, AtALEU, which contains a vacuolar targeting signal, NPIR, in the N-terminal propeptide, but not a precursor of another cysteine proteinase, RD21, which contains no NPIR sequence. Subcellular fractionation revealed that that the AtALEU precursor was associated with PV72-HDEL in the ER of the transgenic plants. To clarify the interaction between the receptor and the ligand, they expressed the luminal domain of PV72 (rPV72) in the insect cells and analyzed their ability to bind the NPIR-containing propeptide of the AtALEU precursor by affinity chromatography and surface plasmon resonance. rPV72 bound to the propeptide with a KD value of 0.1μM, which was low enough for it to function as a receptor. The association of rPV72 with the NPIR-containing propeptide was stabilized in the presence of 1 mM CaCl2. rPV72 bound to the ligand even at pH 4.0 in the presence of Ca2+. Deletion of three repeats of EGF-like motifs of the luminal domain reduced the affinity (KD value of 1.2 μM) between the rPV72 Δ1,2,3 and the propeptide. Overall results suggest that PV72 homolog（s) function as a sorting receptor for the NPIR-containing protease AtALEU to be transported to the lytic vacuoles and that the receptor-mediated transport is regulated by Ca2+ concentration rather than the environmental pH