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.

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.

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.

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.

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).]

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.

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.