The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana

Abstract

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.

Figure 1

Binding of AtELP to vacuolar targeting signals is both pH-dependent and sequence-specific. A, SDS-PAGE and Western analysis of AtELP binding to the Wt-NTPP–targeting propeptides of barley Aleu and Spo. Binding assay with peptide affinity columns was carried out as described in Materials and Methods. The bound proteins were eluted under acidic conditions (pH 4). Fractions were collected for: T, total extract loaded onto each affinity column; FT, flow through; Wash, washes with binding buffer; Elution, protein eluted under low pH (4.0). B, Binding of AtELP to the NTTPs is sequence-specific. The specificity of AtELP binding to the Wt-NTPP-barley Aleu and Wt-NTPP-Spo propeptides was determined in a competition assay. The extract in A was bound to either the Wt-NTPP-barley Aleu or Wt-NTPP-Spo affinity column as described in A. The bound proteins were eluted using increasing concentrations (1–1,000 μM) of the indicated peptides as competitors in the same binding buffer. For each of the flow through (FT), wash, and elution steps, fractions were collected. The indicated fraction numbers for both A and B were analyzed by immunoblotting with α-AtELP antiserum.

Figure 2

Western analysis of BL and Spo in transgenic Arabidopsis root tissue. Total proteins from roots of 2-wk-old seedlings were isolated and analyzed by SDS-PAGE, followed by immunoblotting using the α-BL or α-Spo antiserum. Polypeptides of 18 kD and 27 kD corresponding to the BL and Spo proteins, respectively, were recognized by the corresponding antiserum.

Figure 3

AtELP colocalizes with NTPP-Spo, but not with BL-CTPP in transgenic Arabidopsis roots. Immunogold labeling of BL (A) and Spo (B). Ultrathin sections of transgenic Arabidopsis roots expressing BL or Spo were treated with α-BL (A) or α-Spo (B) antiserum and the bound antibodies were visualized with protein A coupled to gold particles. Arrows represent 15-nm BL- or 10-nm Spo-associated gold particles. C–F, In double immunogold labeling experiments of AtELP and Spo or BL, thin sections of transgenic Arabidopsis roots expressing Spo (C and D) or BL (E and F) were first treated with α-AtELP antisera. The bound antibody was visualized with biotinylated goat α-rabbit IgG and then by streptavidin conjugated to 10-nm gold. After a second fixation step and blocking with an excess of BSA, the sections were treated with α-Spo or α-BL antiserum. The bound antibody was visualized with protein A conjugated to 15-nm gold. Large arrows indicate the 15-nm–labeled α-Spo or α-BL antibody and small arrows indicate the 10-nm–labeled α-AtELP antibody in each case. The sections shown in plates C, E, and F reveal colocalization of AtELP and Spo in the Golgi apparatus (G), and structures near the vacuole (V), but no colocalization with BL. The predominant staining of Spo or BL was found in the vacuole (D and F, respectively). Bars, 0.1 μm.

Figure 4

AtALEU is localized in the vacuole in Arabidopsis. Vacuoles isolated from protoplasts and examined for the presence of AtALEU. A, Protoplasts were prepared by enzymatic digestion of cell suspension culture. B, Vacuoles stained with neutral red were isolated from protoplasts by centrifugation on a discontinuous Ficoll step gradient. Stained vacuoles were visualized by light microscopy. Bars, 50 μm. C, Graphical representation of the ratio of vacuolar enzyme activities U/μg of protein in purified vacuoles, compared with protoplasts. Enzyme activities of two vacuole-specific enzyme markers were determined in protoplasts from cell suspension culture and vacuoles purified from the same protoplasts. The activities were calculated in mol/liter of methylumbelliferone released per hour per microgram of protein. The ratio of the activity of vacuoles with respect to protoplasts was compared (with protoplasts = 1). D, Immunodetection of AtALEU in root (Rt) and cell suspension culture (CS) indicate the presence of a 29-kD polypeptide recognized by the α-AtALEU antiserum. E, Enrichment of AtALEU in vacuoles. Protein extracts prepared from protoplasts (P) and vacuoles (V) of cell suspension culture (A and B) were analyzed by immunoblotting for the presence of AtALEU, and compared with markers for the Golgi apparatus (AtELP) and ER (AtSEC12). F, Immunogold-labeling of AtALEU in Arabidopsis root tissues. Ultrathin cryosections of roots were treated with affinity-purified α-AtALEU antiserum, and the bound antibody was visualized with protein A coupled to 15-nm gold. The arrow indicates the AtALEU-associated 15-nm gold particles. G, AtALEU and Spo are localized in the same vacuoles. In double-immunogold–labeling experiments of AtALEU and Spo, ultrathin cryosections of transgenic Arabidopsis roots expressing Spo were first treated with α-AtALEU antisera. The bound antibody was visualized with biotinylated goat α-rabbit IgG and then by streptavidin conjugated to 15-nm gold. After a second fixation step and blocking with an excess of BSA, the section was treated with α-Spo antiserum and the bound antibody was visualized with protein A conjugated to 10-nm colloidal gold. The large arrow indicates the 10-nm–labeled α-Spo antibody and small arrows indicate the 15-nm–labeled α-AtALEU antibody. Bars, 0.1 μm.

Figure 5

AtELP interacts with the NTPP of AtALEU in a pH-dependent and sequence-specific manner. A, The Wt- and Mt-NTPP-AtALEU peptide sequences used in this experiment are shown. These sequences correspond to the predicted NTPP of AtALEU (Wt-NTPP-AtALEU) or a mutated version of this peptide (Mt-NTPP-AtALEU). B, SDS-PAGE and Western analysis of AtELP binding to the Wt-NTPP– and Mt-NTPP–targeting propeptides of AtALEU. Binding assay with peptide affinity columns were identical to that used in Fig. 1 A. C, Binding of AtELP to the NTPP-AtALEU propeptide is sequence-specific. The specificity of AtELP binding to the Wt-NTPP-AtALEU propeptide was determined in a competition assay identical to that described in Fig. 1 B using increasing concentrations (1–1,000 μM) of the indicated peptides as competitors in the same buffer to elute AtELP bound to the Wt-NTPP-AtALEU peptide column. The Wt-NTPP-barley Aleu and Wt-NTPP-AtALEU peptides bind to the same site on AtELP. AtELP bound to the Wt-NTPP-barley Aleu peptide column can be eluted with increasing concentration of the Wt-NTPP-AtALEU peptide in the same buffer at pH 7 in the competition assay. For each of the flow through (FT), wash, and elution steps, fractions were collected, and the indicated fraction numbers were analyzed by immunoblotting with α-AtELP antiserum as in Fig. 1 B.