Zeolin. A New Recombinant Storage Protein Constructed Using Maize γ-Zein and Bean Phaseolin

Abstract

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of γ-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type γ-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the γ-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.

Protein Quality Control Mechanisms and Protein Storage in the Endoplasmic Reticulum. A Conflict of Interests?

“…These protein bodies sequester a proportion of BiP, which can be released by ATP in vitro treatment, indicating that BiP is not trapped but is acting as a chaperone (heat shock-70 chaperones are ATPases and use ATP hydrolysis to perform their function). Zeolin protein bodies are insoluble and can be solubilized and disassembled by reducing agents (Mainieri et al, 2004). These results indicate that disulfide bonds are a key factor in zeolin protein body assembly and that the process is extensively assisted by BiP.…”

“…Consistently, when the repeat and linker regions of g-zein are appended to the trimeric vacuolar protein phaseolin, the fusion protein, termed zeolin, is retained in the ER and stably deposited in protein body-like structures ( Fig. 2B; see Mainieri et al, 2004). These protein bodies sequester a proportion of BiP, which can be released by ATP in vitro treatment, indicating that BiP is not trapped but is acting as a chaperone (heat shock-70 chaperones are ATPases and use ATP hydrolysis to perform their function).…”

“…However, some native or artificial storage proteins accumulated as protein bodies in the ER also associate for a long time after synthesis with the plant homologue of the immunoglobulin heavy chain binding protein (BiP), which is the ER member of the heat shock 70 chaperone family (Li et al, 1993; and in this issue Mainieri et al, 2004). Persistent interactions with the ER helpers are therefore not sufficient per se for degradation, and this seems to constitute a second paradox, at least in the cells that accumulate storage proteins in the ER.…”

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

“…Previous evidence of a role for disulfide bonds in Zera polymerization came from zeolin expression studies in tobacco (4). Zeolin, a fusion of phaseolin and a ␥-zein sequence equivalent to Zera, was insoluble and formed PBs in the ER when expressed in tobacco (26). When protoplasts expressing zeolin were treated with 2-ME or when a zeolin devoid of the six Cys residues was expressed in the protoplasts, the solubility of the protein was enhanced, allowing its trafficking along the secretory pathway (4).…”

“…The proline-rich N-terminal sequence of ␥-zein comprising the first 112 amino acids (the Zera sequence) (23) fused to a variety of reporter and therapeutic proteins, was shown to induce the stable accumulation of the fusion proteins in ER-derived PB-like structures in plant vegetative tissues (24,25). The ability of a similar sequence to redirect phaseolin, a vacuolar storage protein, to the ER compartment of transformed tobacco leaves in which the fusion protein, named zeolin, is retained and accumulated in PBs, has also been demonstrated (26). Interestingly, the PBinducing capacity of Zera was also demonstrated in a variety of other (non-plant) eukaryotic cells, including mammalian, insect, and fungal cells (24).…”