A novel membrane protein that is transported to protein storage vacuoles via precursor-accumulating vesicles

Abstract

A novel protein, MP73, was specifically found on the membrane of protein storage vacuoles of pumpkin seed. MP73 appeared during seed maturation and disappeared rapidly after seed germination, in association with the morphological changes of the protein storage vacuoles. The MP73 precursor deduced from the isolated cDNA was composed of a signal peptide, a 24-kD domain (P24), and the MP73 domain with a putative long alpha-helix of 13 repeats that are rich in glutamic acid and arginine residues. Immunocytochemistry and immunoblot analysis showed that the precursor-accumulating (PAC) vesicles (endoplasmic reticulum-derived vesicles responsible for the transport of storage proteins) accumulated proMP73, but not MP73, on the membranes. Subcellular fractionation of the pulse-labeled maturing seed demonstrated that the proMP73 form with N-linked oligosaccharides was synthesized on the endoplasmic reticulum and then transported to the protein storage vacuoles via PAC vesicles. Tunicamycin treatment of the seed resulted in the efficient deposition of proMP73 lacking the oligosaccharides (proMP73 Delta Psi) into the PAC vesicles but no accumulation of MP73 in vacuoles. Tunicamycin might impede the transport of proMP73 Delta Psi from the PAC vesicles to the vacuoles or might make the unglycosylated protein unstable in the vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by the action of a vacuolar enzyme to form a 100-kD complex on the vacuolar membranes. These results suggest that PAC vesicles might mediate the delivery of not only storage proteins but also membrane proteins of the vacuoles.

Figure 1.

MP73 Is a Peripheral Membrane Protein of Protein Storage Vacuoles of Pumpkin and Castor Bean. Protein storage vacuoles from pumpkin seed (lane 1), the vacuolar membranes (lane 2), and the alkaline-washed membranes (lane 3) were subjected to SDS-PAGE with Coomassie blue staining (CBB). Five major proteins, MP23, MP27, MP28, MP32, and MP73, are indicated. MP73, MP32, and MP27 were extracted with alkali, whereas two αTIPs, MP23 and MP28, were not (lane 3). Immunoblots with preimmune serum (lane 4) and anti-MP73 antibodies (lanes 5 to 11) are shown. The lanes show the membranes of protein storage vacuoles of pumpkin (lane 4, 50 μg of total proteins; lane 5, 1 μg of total proteins), the membranes of protein storage vacuoles of castor bean (lane 6, 1 μg of total proteins), and each organ of pumpkin (50 μg of total proteins): maturing seed (lane 7), 10-day-old seedlings (lane 8), roots (lane 9), stems (lane 10), and mature leaves (lane 11). 7S, 7S globulin; 11S, 11S globulin.

Figure 2.

Molecular Characterization of MP73. (A) Deduced primary sequence of a precursor of MP73 (preproMP73). The mature MP73 domain is shaded. A Glu/Arg-rich domain of the C-terminal half of MP73 is indicated by darker shading. The determined N-terminal sequence of MP73 is double underlined, and the determined internal sequences are underlined. Open triangle, putative cleavage site of a signal peptide; closed triangle, post-translational processing site; dots, possible glycosylation sites. (B) Hydropathy profile of preproMP73 and its domain structure. A Glu/Arg-rich domain indicated by darker shading is extremely hydrophilic. SP, putative signal peptide; P24, 24-kD domain. (C) Homology plot of preproMP73 showing that the Glu/Arg-rich domain of the C-terminal half is composed of homologous repeat sequences. (D) Alignment of the 13 homologous repeats in the Glu/Arg-rich domain.

Figure 3.

MP73 Is Localized on the Membranes of Protein Storage Vacuoles and ProMP73 Is Localized on the Membranes of the PAC Vesicles in Maturing Pumpkin Seed. (A) Immunocytochemistry with anti-MP73 antibodies showing gold particles on the membrane of a protein storage vacuole. LB, lipid body. Bar = 250 nm. (B) Immunocytochemistry with anti-MP73 antibodies showing gold particles on the membrane of a PAC vesicle. Bar = 250 nm. (C) Immunocytochemistry with anti-MP73 antibodies showing gold particles on the membrane of a PAC vesicle but not on a Golgi complex. G, Golgi complex; Mt, mitochondrion. Bar = 200 nm. (D) Immunoblots of the isolated PAC vesicles and protein storage vacuoles with anti-MP73 antibodies. ProMP73 was detected in the isolated PAC vesicles (lane 1), whereas MP73 was found in the isolated protein storage vacuoles (lane 2, PSV). PSV, protein storage vacuole.

Figure 4.

MP73 Has High Mannose Oligosaccharides but No Complex Glycan. (A) Purified MP73 was incubated in the absence (−) or presence (+) of N-glycosidase F at 37°C for 18 hr and then subjected to SDS-PAGE with Coomassie blue staining. (B) Purified MP73 was incubated in the absence (−) or presence (+) of endoglycosidase H at 37°C for 18 hr and then subjected to SDS-PAGE with Coomassie blue staining. (C) Purified MP73 (lanes 1 and 2) and purified PV72, a vacuolar sorting receptor of pumpkin (lanes 3 and 4), were subjected to SDS-PAGE followed by either staining with Coomassie blue (CBB) or immunoblotting with anti–complex glycan antibodies (anti-CG). MP73ΔΨ, unglycosylated MP73.

Figure 5.

PAC Vesicle–Mediated Transport of ProMP73 and the Effect of Tunicamycin on Transport. (A) Maturing castor bean seed were pulse labeled with ³⁵S-methionine for 30 min and then chased with unlabeled methionine for 0, 3, or 18 hr. Each subcellular fraction of the labeled tissues was immunoprecipitated with anti-MP73 antibodies, and the immunoprecipitates were subjected to SDS-PAGE and fluorography. (B) Maturing castor bean seed were incubated without tunicamycin (lane 1) or in the presence of 3 μg/mL (lane 2) or 30 μg/mL (lane 3) tunicamycin for 3 hr before a 4-hr pulse labeling with ³⁵S-methionine. Each homogenate was immunoprecipitated with anti-MP73 antibodies, and the immunoprecipitates were subjected to SDS-PAGE and fluorography. In vitro translation products directed by poly(A) RNA from the maturing seed also were immunoprecipitated to show the preproMP73 (lane 4). (C) Maturing castor bean seed were incubated in the absence (−) or presence (+) of 30 μg/mL tunicamycin for 3 hr before a 4-hr pulse labeling with ³⁵S-methionine. The labeled seed were applied to subcellular fractionation. Immunoprecipitates of each fraction with anti-MP73 antibodies were subjected to SDS-PAGE and fluorography. S,13% (w/w) sucrose fraction and the 13 to 20% (w/w) interface fraction containing vacuolar membranes; E, ER fraction; P, PAC vesicle fraction; C, crystalloids of protein storage vacuoles; proMP73ΔΨ, unglycosylated form of proMP73.

Figure 6.

In Vitro Conversion of ProMP73 to MP73. PAC vesicles from the pulse-labeled maturing castor bean seed were incubated with an extract of protein storage vacuoles at 30°C for 0 or 16 hr, followed by immunoprecipitation with anti-MP73 antibodies. The immunoprecipitate was subjected to SDS-PAGE and fluorography. ProMP73 was converted to MP73.

Figure 7.

Oligomeric Structure of MP73. The membranes of protein storage vacuoles were treated with 0, 0.125, and 0.250% DSP. Aliquots of the reaction mixture were subjected to SDS-PAGE under nonreducing (lanes 1 to 3) or reducing (lanes 4 to 6) conditions with 2-mercaptoethanol (2ME) and subsequently were subjected to immunoblotting with anti-MP73 antibodies. A 100-kD complex was detected on the blot only under nonreducing conditions (lanes 2 and 3). The molecular mass of each marker protein is given at right in kD. The asterisk indicates degradation products of MP73.

Figure 8.

Developmental Changes in the Level of MP73 in Pumpkin Cotyledons during Seed Maturation, Germination, and Subsequent Seedling Growth. (A) Pumpkin cotyledons were harvested 18, 19, 21, 24, 26, 31, and 38 days after anthesis (Seed maturation) and 0, 2, 4, and 6 days after germination (Seed germination). The homogenate from each 75 μg (fresh weight) of the cotyledon was subjected to SDS-PAGE with