Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm

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.

Divergent properties of prolamins in wheat and maize

“…The morphology of the protein bodies of Dx5 transgenic endosperm is similar to that in wheat or barley, in a way that protein bodies are interspersed with a fibrillar matrix Arcalis et al 2004). Beyond normal zein protein bodies, smaller protein bodies occur with the presence of Dx5.…”

“…In spite of the divergence in seed storage proteins, however, prolamins from all species accumulate similar ER-derived protein bodies, suggesting that the basic functional interactions required for protein body formation have been preserved across species. Understanding the roles of different prolamins in protein body formation may allow a rational design of cereal products with improved functionality and the production of foreign proteins (Arcalis et al 2004).…”

Increasing the production yield of recombinant protein in transgenic seeds by expanding the deposition space within the intracellular compartment

“…These recombinant proteins are mainly or exclusively deposited into the ER or ER-derived PBs in transgenic cereal seeds, although accumulation sometimes occurs in the PSV instead, due to an interaction between the recombinant protein and endogenous seed proteins. 32,33 When the 7Crp antigen peptide derived from the major T-cell epitopes from Japanese cedar pollen allergens and other types of cytokines, such as IL-4 and IFN-r, were expressed in conjunction with the simultaneous suppression of prolamin genes deposited in ER-derived PB-I, 34 these recombinant proteins were produced at high levels, as was also observed with the production of hIL-10, confirming the efficacy of this technique as a general strategy for increasing the production of recombinant proteins (Fig. 1).…”

Vacuolar Sorting Behaviors of 11S Globulins in Plant Cells

“…It has been reported that intracellular deposition is tissue and plant species dependent. [27][28][29] The difference in plant cell types also might affect the presence of protein aggregates. We should examine the differences in character depending on plant cell types to determine the membrane traffic of 11S.…”