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. 2004 Nov;136(3):3457-66.
doi: 10.1104/pp.104.050153. Epub 2004 Oct 15.

Unexpected deposition patterns of recombinant proteins in post-endoplasmic reticulum compartments of wheat endosperm

Elsa Arcalis  1 , Sylvain Marcel, Friedrich Altmann, Daniel Kolarich, Georgia Drakakaki, Rainer Fischer, Paul Christou, Eva Stoger
Affiliations

Unexpected deposition patterns of recombinant proteins in post-endoplasmic reticulum compartments of wheat endosperm

Elsa Arcalis et al. Plant Physiol. 2004 Nov.

Abstract

Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals.

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Figures

Figure 1.
Figure 1.
Wheat protein bodies. A, Light microscopy. Cross-section of a wild-type seed. Spurr semithin section, stained with methylene blue. Wheat protein bodies (arrow heads) can be seen in the large, central vacuole (v) of cells in the first layers of the endosperm (E). B, Light microscopy. Cross-section of wild-type seed. Spurr semithin section, stained with methylene blue. The protein bodies are sequestered in the central vacuole (arrow head) of the endosperm cell. C, Confocal microscopy. 3-D reconstruction from optical confocal sections, showing the localization of HMW glutenins in wild-type seed. A protein body can be seen within the vacuole. Note the aggregation of prolamin bodies and the presence of individual prolamin bodies close to the aggregate (arrows). Inclusion bodies can be seen on the surface of the aggregated prolamin bodies (arrow heads) D, Electron microscopy. Spurr thin section, general nonspecific staining, wild-type seed. Prolamin bodies (pr) are shown with peripheral inclusion bodies (I). Membranes can be seen surrounding the protein body (arrows) and between two prolamin bodies (double arrow). E and F, Electron microscopy. Spurr thin section, detection of unsaturated lipids, wild-type seed. A fine precipitate can be seen on the outer protein body membrane (double arrows). Note the continuity of the protein body membrane over the inclusion bodies (arrow heads), and the absence of membrane between the prolamin (pr) and the inclusion bodies (I, arrows). Myelin-like structures are indicated with an asterisk. Bars, 500 μm (A and B); 50 μm (C); 0.5 μm (D–F). A, aleurone; s, starch grains.
Figure 2.
Figure 2.
Localization of recombinant HSA. A, Fluorescence microscopy. Spurr semithin section. Strong labeling can be seen in the protein bodies (arrows). Note the labeling in several small, individual prolamin bodies. B and C, Confocal microscopy. Tangential (B) and cross-optical (C) sections from wheat leaves. Note the labeling inside the cell. Both the vacuole (v) and the apoplast (arrow heads) are label-free. D, Confocal microscopy. 3-D reconstruction from optical confocal sections. HSA accumulates in the prolamin body. Note the nonlabeled inclusion bodies (arrow heads). E, Abundant gold probes in the prolamin bodies (pr), nonsignificant labeling in the inclusion bodies (arrow heads). Note the membrane between prolamin bodies (double arrow). F, Cytosolic prolamin body (pr) decorated with abundant gold probes. Arrow heads indicate two rough ER cisternae next to it. G–J, Confocal microscopy, optical sections from the protein body in (D). The inclusion bodies are indicated with an arrow head. Those inclusion bodies appear on the surface of the prolamin body (D, G–I) or embedded within it (J). Bars, 500 μm (A); 200 μm (B and C); 100 μm (D); 0.5 μm (E); 0.25 μm (F), and 200 μm (G–J). a, Aleurone; E, endosperm.
Figure 3.
Figure 3.
Localization of recombinant pea legumin. A, Confocal microscopy. 3-D reconstruction from optical confocal sections. Pea legumin is deposited within the inclusion bodies (arow heads). B, Optical section from the protein body in (A). Pea legumin is located in the inclusion bodies (arrow heads), which are distributed around two prolamin bodies (pr). The prolamin bodies (pr) are difficult to see in this figure. Bars, 200 μm.
Figure 4.
Figure 4.
Localization of recombinant phytase. A, Fluorescence microscopy. LR White semithin section. Phytase accumulates in the protein bodies (pb). Note the absence of a signal in the apoplast (arrows). B and C, Electron microscopy. LR White thin sections. Significant labeling is seen in the prolamin bodies (pr), but none is found in the inclusion bodies (arrow heads). C, Single protein body with a triticin inclusion (arrow head). Bars, 500 μm (A); 0.25 μm (B and C). s, Starch.
Figure 5.
Figure 5.
Immunoblot analysis. A, Detection of HSA in endosperm extracts from single seeds. Lanes 1 and 7, Nontransformed seeds; lanes 2 to 6, transgenic seeds containing HSA. Detection with anti-HSA antibody (lanes 1–5) or with anti-KDEL antibody (lanes 6 and 7). B, Detection of partially purified phytase from seeds. Lanes 1 (control; nontransformed seed) and 2 (transgenic seed), antiphytase antiserum. Lanes 2 (transgenic seed) and 3 (control; nontransformed seed), detected with Aleuria aurantia lectin.
Figure 6.
Figure 6.
Glycoproteomic analysis of endosperm-derived phytase. The upper section presents the sequence of phytase with identified tryptic peptides shown in bold and underlined; the signal peptide is shown in italic; and potential glycopeptides after tryptic digestion are underlined in gray, with potential N-glycan binding sites marked with arrows. Most of the nonglycosylated peptides could be identified by peptide mass fingerprinting ensuring the protein was correctly identified. In addition, the glycopeptide (121–145) was found to contain either MMXF or MUXF glycans (spectrum not shown). The lower section shows the MALDI-TOF mass spectrometry spectrum acquired from the enzymatically released and isolated N-glycans. MMXF was identified as the major N-glycan structure, but further complex and oligomannosidic structures were also identified. N-glycans were identified mainly as their sodium adducts. (•), GlcNAc; (○) Man; (▴) Fuc; (▪) Xyl. Key to monosaccharide linkage is located in the upper right corner of the lower section.
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