Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana

Abstract

The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1/AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 12S globulin in a Ca²⁺-dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.

The seeds of higher plants accumulate large quantities of seed proteins including storage proteins and lectins (1). These seed proteins are localized in two different compartments. One is protein bodies for zeins and prolamins that are directly derived from endoplasmic reticulum in maize and rice endosperm (2). Another is protein storage vacuoles (PSVs) for globulins and albumins that are a special type of vacuole (3). During seed maturation, the precursors of globulins and albumins are synthesized on rough endoplasmic reticulum and are sorted to PSVs (4, 5), where they are converted into the mature forms and accumulated (6-8). Vacuolar targeting signals for the seed proteins have been found in Brazil nut 2S albumin (9), common bean phaseolin (10), castor bean ricin (11), and soybean β-conglycinin α′ subunit (12). Vacuolar sorting of phaseolin has been shown to be saturated in overexpression experiments (13). These results have suggested a receptor-mediated transport of the storage proteins. Despite many investigations, however, the sorting machinery for storage proteins has not been determined.

We previously showed that unique vesicles, precursor-accumulating vesicles, mediate the mass transport of storage proteins to PSVs in maturing pumpkin seeds (14). Isolated precursor-accumulating vesicles contain a large amount of the precursors of major storage proteins. On the membrane of the precursor-accumulating vesicles, we found PV72, a type I integral membrane protein composed of a large luminal domain and a transmembrane domain followed by a short cytosolic tail that contains a potential tyrosine-based motif (15). PV72 is the next most abundant protein after the storage protein precursors in the precursor-accumulating vesicles. PV72 is specifically and transiently accumulated in maturing pumpkin seeds in association with the synthesis of storage proteins, but is not expressed in vegetative tissues (16). PV72 has been shown to bind to peptides derived from pumpkin 2S albumin (15, 17) and also to the precursor of pumpkin 2S albumin (16) in the presence of Ca²⁺. These observations imply that PV72 functions as a vacuolar sorting receptor (VSR) for storage proteins in pumpkin seeds.

Proteins homologous to pumpkin PV72 have been found in various plants. These include pea BP-80 (18), Arabidopsis AtELP (19), and Vigna VmVSR (20). AtELP is localized in the Golgi complex and prevacuolar compartments (21, 22). AtELP bound in vitro to the propeptides of both Arabidopsis aleurain, AtA-LEU, and sweet potato sporamin (22). The propeptides contain an Asn-Pro-Ile-Arg (NPIR) sequence, a typical vacuolar targeting signal, for lytic vacuoles. In transgenic Arabidopsis plants expressing sporamin, AtELP was colocalized with sporamin at the Golgi complex (22). The authors suggested that AtELP functions as a sorting receptor for NPIR-containing proteins in the lytic vacuoles. Recently, Larval et al. (23) reported that seed germination was blocked in AtELP-antisense Arabidopsis plants. BP-80 also bound in vitro to the peptides with an NPIR sequence (24). BP-80 sorted the GFP fusion protein with an NPIR-containing propeptide of petunia aleurain into the vacuoles of yeast (25). BP-80 is localized in clathrin-coated vesicles and the Golgi complex of maturing pea cotyledons (26, 27). Robinson and Hinz (28) reported that storage proteins of maturing pea cotyledons were transported by dense vesicles rather than clathrin-coated vesicles. Therefore, it is thought that BP-80 is not involved in the sorting of storage proteins into PSVs.

Arabidopsis has seven homologs of PV72, which we designated AtVSR1-AtVSR7 (VSR of Arabidopsis thaliana). The homolog closely related to PV72 was designated AtVSR1, which is also referred to as AtELP. To explore the function of each receptor, we used a reverse-genetic approach to identify a series of Arabidopsis mutants, each of which is defective in one of these receptor genes. Here, we report the characterization of atvsr1 mutants in which AtVSR1 gene was disrupted by the insertion of transfer DNA (T-DNA). Our observations indicate that AtVSR1 function as a sorting receptor for storage proteins in maturing seed cells.

Materials and Methods

Plant Materials and Growth Conditions. A. thaliana ecotype Columbia (Col-0), Wassilewskija-2 (Ws-2), and Lansberg er (Ler) were used. Seeds of Arabidopsis were surface-sterilized and then sown on soil or onto 0.5% Gellan Gum (Wako, Tokyo) that contained 1% sucrose and Murasige-Skoog medium. After a 2-day incubation at 4°C to break seed dormancy, WT and the atvsr1-1 mutant plants were grown side by side at 22°C under continuous light.

Screening for T-DNA Insertion Mutants. To isolate AtVSR knockout mutants, we screened the collection of T-DNA mutagenized Arabidopsis plants of the Arabidopsis Knockout Facility of Wisconsin University (29). In the screening by PCR, we used a set of the unique primers for AtVSR1/AtELP (the forward primer 5′-AGAACAGCCAATTGAAGCAAGCATTTGTG-3′ and the reverse primer 5′-CACATGAGGCTTTTCCAAGAGTCGCAGAA-3′) and the left border primer JL-202 of T-DNA. Similarly we isolated the mutants, atvsr3-1, atvsr4-1, atvsr5-1, and atvsr6-1, with each set of the forward and reverse unique primers, respectively; 5′-CCAAACCGCTCAACAAGAAAAACCAGATT-3′ and 5′-TCGTTAAGGGCCACATCAGTTAAAAGAAC-3′ for AtVSR3; 5′-CGTCGTCGATATCTGTTTGACTTTGAATA-3′ and 5′-CAGAAGTCACATGGACACAATTGACAAGA-3′ for AtVSR4; 5′-TCACATGGCTCGTGTGGGGTTGTATTTGA-3′ and 5′-CAATCTTCATTTGAGATGACAAGAGTTCC-3′ for AtVSR5; and 5′-CCATACCTTTTTAACGTTTTGCCACTTGA-3′ and 5′-TGAAAAGAGTCAATAAAAATGACCCAAAA-3′ for AtVSR6. Seeds of the atvsr1-1, atvsr3-1, atvsr4-1, atvsr5-1, and atvsr6-1 mutants (Ws-2) were donated by the Arabidopsis Biological Resource Center (Ohio State University, Columbus). Seeds of atvsr1-2 (Col-0) were donated by the Max Planck Institute for Plant Breeding Research (Cologne, Germany). Seeds of the atvsr7-1 mutant (Col-0) were donated by Salk Institute Genome Analysis Laboratory. Seeds of the atvsr2-1 mutant (Ler) were donated by Cold Spring Harbor Laboratory. To determine the T-DNA insertion site in the mutants, we cloned and sequenced the DNA fragments flanking the T-DNA insertion site. The atvsr1-1 mutant has the T-DNA insertion at 1,364 nt downstream from the initiation codon, which is located in the third exon of AtVSR1. The atvsr1-2 mutant has the T-DNA insertion at 2,606 nt downstream from the initiation codon, which is located in the seventh intron of AtVSR1.

SDS/PAGE and Immunoblot Analysis. Protein extracts were prepared from dry seeds or seedlings in SDS/PAGE sample buffer. Samples were subjected to SDS/PAGE followed by either Coomassie blue staining or blotting to Immobilon-P membranes (Nihon Millipore, Tokyo). The membranes were treated with Abs against AtELP (1:2,000; Rose Biotechnology, Palo Alto, CA), Arabidopsis 2S albumin (1:10,000), Arabidopsis 12S globulin α-subunit (1:10,000) or barley aleurain (2F5, 1:5,000) (30). Abs against Arabidopsis 2S albumin and 12S globulin α-subunit were described (7). Immunoreactive signals were detected with an enhanced chemiluminescence detection system (ECL, Amersham Biosciences).

Transformation of atvsr1-1 Mutant with AtVSR1 cDNA. A full-length cDNA for AtVSR1 (YAY356, pAtVSR1-cDNA) was donated by the Arabidopsis Biological Resource Center (Ohio State University). The promoter (2,000 bp) and terminator (500 bp) regions of AtVSR1 gene were amplified by PCR with Arabidopsis genomic DNA and cloned into pT7Blue (Novagen) to make pP-AtVSR1 and pT-AtVSR1, respectively. The specific primer sets used were 5′-CGTTCATGGCTAAAACCGTTTGGGAACCTT-3′ and 5′-TGAAATCGAACCTCCACGATCGATAAGGAC-3′ for the promoter and 5′-TCCATGTAAATGAATCAATGGACTTGATAC-3′ and 5′-GAGCTCACTAGTAACTCCAAACACATATTG-3′ for the terminator. pP-AtVSR1-cDNA-T, which contains a chimeric gene composed of the promoter, cDNA-coding region and terminator of AtVSR1, was generated with pP-AtVSR1, pAtVSR1-cDNA, and pT-AtVSR1. The chimeric gene was then introduced into binary Ti vector pGreenII 0229 (31) to produce pG_pAtVSR1-cDNA. pG_pAtVSR1-cDNA was introduced into Agrobacterium tumefaciens (strain EHA105) by electroporation and then into the atvsr1-1 mutant. T1 plant was selected for resistance to bialaphos (75 mg/liter). The presence of the transgene pAtVSR1-cDNA was tested by PCR by using specific primers, 5′-CTATGTGACTGACTTTGCTATCCGGTGTCC-3′ (F1) and 5′-GATAGCTGGTTCCGTTGACTCTTGAAAACC-3′ (R1). Both genomic DNA and whole proteins were prepared from T2 seeds with a solution of 50 mM Tris·HCl (pH 6.8), 250 mM NaCl, 25 mM EDTA, 1% SDS, 0.5% 2-mercaptoethanol, and 5% glycerol.

Microscopic Analysis. For immunoelectron microscopy, we fixed, dehydrated, and embedded the dry seeds in LR white resin (London Resin, Basingstoke, U.K.) as described (32), except for using the fixative containing 10% dimethyl sulfoxide. Samples were treated with Abs against Arabidopsis 2S albumin (dilution 1:50) and Arabidopsis 12S globulin (dilution 1:50). Sections were examined with a transmission electron microscope (1200EX; JEOL, Tokyo) at 80 kV.

For laser-scanning confocal microscopy, dry seeds from WT and the atvsr1 mutant plants were pressed in glycerol between slide-glass and cover-strip and to push out cotyledons. They were inspected with a fluorescence microscope (Axioplan 2, Zeiss) equipped with a confocal laser-scanning unit (CSU10, Yokogawa Electronic, Tokyo) and the laser unit (Sapphire 488, Coherent Radiation, Palo Alto, CA). We used the filter set for GFP fluorescence (510AF23 Omega Optical, Brattleboro, VT). Autofluorescent images of PSVs were acquired by a CCD camera (OrcaER, Hamamatsu Photonics, Hamamatsu City, Japan) and processed by the IPLAB software (Scanalytics, Fairfax, VA) and PHOTOSHOP 5.5 (Adobe Systems, Tokyo).

Affinity Column Chromatography. Affinity column chromatography was carried out essentially as described (17). We prepared affinity columns conjugated with the C-terminal peptide (PASYGRPRVAAA) of Arabidopsis 12S globulin isoform 4 (CruA). The synthesized peptide (5 mg) was immobilized to Aminolink coupling gel (2 ml of a bed volume; Pierce). Protein extracts from dry seeds of Arabidopsis were applied to the column that had been equilibrated with a Hepes buffer (20 mM Hepes-NaOH, pH7.0/150 mM NaCl/1% CHAPS) containing 1 mM CaCl₂. The bound molecules were eluted with the Hepes buffer containing 5 mM EGTA or 25 mg/ml of each peptide: ASYGRPRVA A A derived from 12S globulin, ANIGFDESNPIRMVSDGLREV derived from AtALEU, and PSRCNLSPMRCPMGGSIAGF derived from Brazil nut 2S albumin. Each fraction was subjected to immunoblotting with anti-AtELP Ab. AtVSR1-L gave a weak signal on the blot of the total extract from the seeds probably because of less solubility or less stability of AtVSR1-L.

Results and Discussion

Identification of an AtVSR1 Knockout Mutant. We isolated Arabidopsis mutants in which each gene homologous to pumpkin PV72 was disrupted by an exogenous T-DNA. Fig. 1A shows a phylogenetic tree indicating the relationship between PV72 (15) and homologs, including At3g52850/AtVSR1/AtELP/atbp80b (19, 33), pea BP-80 (18), and black gram VmVSR (20). In immunoblot analysis, Ab against AtELP reacted with two bands in protein from WT seeds, corresponding to molecular masses of ≈80 and 60 kDa (Fig. 1B, WT). The 60-kDa band might be derived from the 80-kDa band. Seeds of the atvsr1-1 mutant (Ws-2) lacked the 60-kDa band (AtVSR1-S) and the 80-kDa band (AtVSR1-L), as shown in Fig. 1B (atvsr1-1). A faint signal at 80 kDa on the blot of the atvsr1-1 mutant was due to the cross-reactivity of the Ab with other AtVSRs. Seeds of the other mutants, atvsr2-1 (Ler), atvsr3-1 (Ws-2), atvsr4-1 (Ws-2), atvsr5-1 (Ws-2), and atvsr6-1 (Ws-2), produced two bands, like the WT (Ws-2) seeds. AtVSR1-S was less abundant than AtVSR1-L in WT (Col-0) and atvsr7-1 (Col-0) seeds. We focused on the atvsr1-1 mutant, which has a T-DNA insertion in the third exon of the AtVSR1 gene (Fig. 1C).

Fig. 1.

VSR proteins and Arabidopsis VSR knockout mutants. (A) A phylogenetic tree of VSR proteins of Arabidopsis, pumpkin, pea, and black gram. The region between Asp-176 and Gly-508 of PV72 was aligned with the corresponding regions of the other proteins. The tree was drawn with the clustalw and treeview programs. The scale represents the evolutionary distance expressed as the number of substitutions per amino acid. Arabidopsis has seven VSR genes. The homolog closely related to PV72 was designated AtVSR1, which is also referred to as AtELP. (B) Immunoblot analysis of dry seeds from Arabidopsis VSR knockout mutants (atvsr1-1-atvsr7-1) with Ab directed against AtELP. The WT seeds (Ws-2 and Col-0) produced two bands: AtVSR1-L and AtVSR1-S. (C) A schematic representation of the AtVSR1 gene and the positions of the T-DNA insertions in the atvsr1-1 and atvsr1-2 alleles. Open boxes represent exons, and solid lines represent introns. F1 and R1, a primer set used for PCR in Fig. 3B.

Abnormal Accumulation of Storage Protein Precursors in atvsr1 Seeds. We attempted to clarify the effect of AtVSR1 deficiency on the quality or quantity of the storage proteins in dry seeds. Fig. 2A shows a comparison of the seed storage protein profiles of WT and atvsr1-1 seeds (five grains) on an SDS gel with Coomassie blue staining. The WT seeds accumulated the α- and β-subunits of 12S globulin and the large and small subunits of 2S albumin (Fig. 2 A, WT). Interestingly, atvsr1-1 seeds abnormally accumulated 17-, 49-, 51-, and 54-kDa proteins in addition to the mature storage proteins (Fig. 2 A, atvsr1-1). The atvsr1-1 seeds accumulated a smaller amount of mature 12S globulin and a similar amount of 2S albumin compared to the WT seeds. Immunoblot analysis showed that the additional proteins found in atvsr1-1 seeds were the precursors of storage proteins (Fig. 2B, atvsr1-1). The accumulation of the precursor proteins in the atvsr1-1 seeds indicated some defect in the intracellular transport of storage proteins in the mutant cells. This phenotype of atvsr1-1 was recessive. Seeds of the other mutants (atvsr2-1, atvsr3-1, atvsr4-1, atvsr5-1, atvsr6-1, and atvsr7-1) did not accumulate storage protein precursors (data not shown).

Fig. 2.

The atvsr1 mutant accumulates large amounts of the precursors of seed storage proteins in dry seeds. (A) Protein profiles of the dry seeds (five grains) of the WT and atvsr1-1 mutant. (B) Immunoblot analysis of the dry seeds of the WT and atvsr1-1 mutant with anti-12S globulin (anti-12S) and anti-2S albumin (anti-2S) Abs. The atvsr1-1 seeds accumulated large amounts of the precursors of 12S globulin (p12S) and 2S albumin (p2S), whereas WT seeds accumulated only the mature forms of 12S globulin (12S) and 2S albumin (2S). The amount of the mature 12S globulin in the atvsr1-1 seeds was lower than that in WT seeds. α and β, 12S globulin subunits; L and S, 2S albumin subunits.

We obtained another atvsr1 allele to verify that the disruption of AtVSR1 gene causes the abnormal accumulation of storage protein precursors in dry seeds. The atvsr1-2 mutant has T-DNA in the seventh intron of the AtVSR1 gene (Fig. 1C). Immunoblot analysis with Abs against storage proteins revealed that atvsr1-2 seeds accumulated storage protein precursors, pro2S albumin and pro12S globulin, as did the atvsr1-1 seeds (Fig. 3A).

Fig. 3.

Confirmation and restoration of the atvsr1 phenotype. (A) Immunoblot analysis of the dry seeds of the WTs (Ws-2 and Col-0), atvsr1-1 mutant, and atvsr1-2 mutants with anti-12S globulin (anti-12S) and anti-2S albumin (anti-2S) Abs. (B) The atvsr1-1 mutant was transformed with pAtVSR1::AtVSR1-cDNA. Shown is a PCR-based analysis of the AtVSR1 locus and the presence of pAtVSR1::AtVSR1-cDNA in the WT, in four independent T2 seeds, and in the atvsr1-1 mutant (Upper). Immunoblot analysis of their dry seeds with anti-12S globulin (anti-12S) and anti-2S albumin (anti-2S) Abs (Lower). p12S, the precursors of 12S globulin; p2S, the precursors of 2S albumin; 12S-α and 2S-L, a 12S globulin subunit and a 2S albumin subunit.

To further confirm that the accumulation of storage protein precursors is caused by the absence of AtVSR1, we expressed AtVSR1 in an atvsr1-1 background to restore the WT phenotype. The atvsr1-1 mutant plants were transformed with AtVSR1 cDNA under the control of the AtVSR1 promoter (pAtVSR1::AtVSR1-cDNA) together with a bar gene providing resistance to bialaphos for the selection of transformants. The presence of the transgene pAtVSR1::AtVSR1-cDNA and storage protein precursors was analyzed with T2 seeds from a bialaphos-resistant plant. Fig. 3B Upper shows the results of PCR-based analysis with four T2 seeds (lines 1-4). Line 1 is the atvsr1-1 mutant, whereas lines 2-4 are atvsr1-1 mutants containing pAtVSR1::AtVSR1-cDNA. Immunoblot analysis with Abs against storage proteins revealed that line 1 contained storage protein precursors, as did the atvsr1-1 mutant (Fig. 3B Lower). In contrast, lines 2, 3, and 4 accumulated no or much less amounts of the precursors, as does the WT (Fig. 3B Lower). These results confirm that AtVSR1 is responsible for the proper accumulation of mature forms of storage proteins in Arabidopsis seeds.

atvsr1 Mutant Missorts Storage Proteins by Secreting Them from Cells. Storage proteins are accumulated in PSVs, a special type of vacuole, in dry seed cells. Fig. 4 A and B shows the morphologies of the cotyledon cells and the autofluorescent PSVs of the WT and atvsr1-1 seeds. The atvsr1-1 seeds have distorted cells and the PSVs are smaller than those of the WT seeds. The average diameters of PSVs (n = 20) are 7.20 ± 0.99 μm for WT and 5.70 ± 0.94 μm for the mutant. This observation suggests that AtVSR1 is involved in the biogenesis of PSVs. The failure in targeting storage proteins to PSVs should cause them to accumulate in the extracellular space. Electron micrographs revealed that the extracellular space of the atvsr1-1 seeds (Fig. 4D) is abnormally enlarged and filled with electron-dense material compared to WT seeds (Fig. 4C). As expected, immunogold analysis of atvsr1-1 seeds indicated that this electron-dense material has high concentrations of the storage proteins, 12S globulin (Fig. 4F) and 2S albumin (Fig. 4H). The storage proteins are distributed in the PSVs and the electron-dense extracellular space in the atvsr1-1 seeds, whereas 12S globulin and 2S albumin are deposited only in the PSVs in WT seeds. These observations indicate that the atvsr1-1 mutant missorts the storage proteins by secreting them from cells, resulting in an enlarged and electrondense extracellular space and the shrinkage of the cells in the seeds (Fig. 4B). The clear reduction in the amount of mature 12S globulin in the atvsr1-1 seeds (Fig. 2 A) may be responsible for the smaller size of the PSVs in the mutant cells (Fig. 4B). These results indicate the involvement of AtVSR1 in the targeting of both 12S globulin and 2S albumin to PSVs in the cells of maturing seeds.

Fig. 4.

The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. Shown is autofluorescence of PSVs in the dry seeds of the WT (A) and the atvsr1-1 mutant (B). The atvsr1-1 seeds have distorted cells and smaller PSVs than those of WT seeds. Ultrastructures of seed cells of the WT (C) and atvsr1-1 mutant (D) are shown. The extracellular space of the atvsr1-1 seeds was abnormally enlarged and filled with electron-dense material (arrowheads in D). (E-H) ImmunoGold analysis with Ab against 12S globulin (E and F) or 2S albumin (G and H). Both storage proteins were distributed in the PSVs and the electron-dense extracellular space of the atvsr1-1 seeds (arrowheads in F and H). CW, cell wall. [Scale bars = 10 μm(A and B), 5 μm(C and D), and 1 μm(E-H).]

AtVSR1 Binds the C-Terminal Peptides of Storage Proteins. The vacuolar-targeting signals of Arabidopsis 12S globulin or 2S albumin are not known. In contrast, the vacuolar-targeting signals of Brazil nut 2S albumin (9), common bean phaseolin (10), and the soybean β-conglycinin α′ subunit (12) were determined to be located at the C termini of the proteins. Therefore, we prepared an affinity column conjugated with the C-terminal peptide of Arabidopsis 12S globulin to clarify the binding ability of AtVSR1 to the storage proteins. Fig. 5 shows that AtVSR1-S specifically bound to the column in the presence of Ca²⁺ and was eluted with EGTA solution. Both Mg²⁺ and Mn²⁺ showed no effect on the binding of AtVSR1-S (data not shown). These results are consistent with our previous results (17). The protein bound to the column was competitively eluted with a solution containing Arabidopsis 12S globulin (At12S). The bound protein was also competitively eluted with a solution containing each of the well-known signals Brazil nut 2S albumin peptide (Be2S) and AtALEU peptide (AtALEU). These results suggest that AtVSR1 binds to storage proteins in a Ca²⁺-dependent manner. It is possible that the C-terminal region of 12S globulin acts as a targeting signal to the PSV.

Fig. 5.

AtVSR1 binds to a C-terminal peptide of Arabidopsis 12S globulin, and the bound AtVSR1 is competitively eluted by known vacuolar-targeting signals. AtVSR1 from the microsomal fraction of WT seeds was bound to an affinity column with At12S peptide in the presence of 1 mM CaCl₂. AtVSR1 was eluted by each of At12S peptide, Be2S peptide, AtALEU peptide, or EGTA. Each fraction eluted from the columns was subjected to SDS/PAGE followed by immunoblot analysis with anti-AtELP Ab.

Lytic Enzymes Are Sorted Normally to Vacuoles in atvsr1 Seedlings. Despite the secretion of storage proteins and their accumulation in the extracellular space, the atvsr1 seeds showed normal germination and growth (data not shown). To clarify the destiny of the storage protein precursors, we examined the protein profiles of the seedlings after germination. The precursors of 12S globulin and 2S albumin were degraded after germination of the atvsr1-1 seeds (Fig. 6A, indicated by arrows in Right). It is likely that some proteinase(s) is secreted to degrade the storage proteins in the extracellular space after germination of the atvsr1-1 seeds. The mature forms of 12S globulin and 2S albumin were also degraded normally in the atvsr1-1 seeds after germination, like those of the WT seeds (Fig. 6A). The degradation of storage proteins is accompanied by the conversion of PSVs into lytic vacuoles. Our observation suggests that proteinases responsible for the degradation of storage proteins are transported normally to the vacuoles of atvsr1-1 seedlings. AtVSR1 does not appear to be responsible for sorting such proteinases to the lytic vacuoles.

Fig. 6.

Developmental changes in the storage proteins and AtALEU in WT and atvsr1-1 seeds after germination. (A) Seeds and seedlings of the WT and the atvsr1-1 mutant at 0-6 days after germination were subjected to SDS/PAGE followed by Coomassie blue staining. p12S, precursors of 12S globulin; p2S, precursors of 2S albumin; α and β, 12S globulin subunits; 2S, 2S albumin. Asterisks indicate RuBisCO located in the chloroplasts. (B) Immunoblot analysis of seeds and seedlings of the WT and the atvsr1-1 mutant at 0-5 days after germination with a mAb (2F5) directed against ALEU, an NPIR-containing vacuolar cysteine proteinase. No AtALEU precursor was detected, suggesting that AtVSR1 is not involved in the vacuolar sorting of AtALEU. m, mature AtALEU; p, predicted position of the precursor of AtALEU.

An in vitro-binding assay demonstrated an interaction between AtVSR1 and the NPIR-containing signal of AtALEU (Fig. 5). This result suggests that AtVSR1 sorts the precursor of AtALEU to the lytic vacuoles. Therefore, we examined the vacuolar sorting of AtALEU, which is newly synthesized and accumulated in the lytic vacuoles after seed germination. Deletion of the NPIR signal caused the secretion of a barley aleurain from cells, where it remained in the precursor form (34). A sorting defect could cause the accumulation of the AtALEU precursor in atvsr1-1 seedlings. However, Fig. 6B shows that no precursor forms of AtALEU accumulated in the atvsr1-1 seedlings. This result suggests that AtVSR1 is not involved in the sorting of AtALEU to lytic vacuoles. Some of the other AtVSRs possibly plays a role in the transport of such lytic enzymes that are targeted to lytic vacuoles.

Our findings indicate that AtVSR1 is a sorting receptor that sorts storage proteins to PSVs. However, some of these storage proteins are still found in the PSVs of mutant seed cells (Fig. 4). This data raises the question of how the receptor-deficient atvsr1 seeds deliver storage proteins to PSVs. We described that storage protein aggregates that are formed within the endoplasmic reticulum are transported directly to PSVs in a Golgi-independent manner (14). It is possible that some of the storage proteins are transported to PSVs in the atvsr1 seeds by this pathway that is driven by receptor-independent machinery. Another possibility is that the other AtVSRs are also involved in the transport of storage proteins together with AtVSR1. Each of the seven AtVSRs possibly functions as a sorting receptor for some vacuolar proteins in those tissues in which it is specifically expressed. Further experiments with the other atvsr mutants should provide valuable insights into the functional differentiation of the AtVSRs.