Two Structural Domains Mediate Two Sequential Events in [gamma]-Zein Targeting: Protein Endoplasmic Reticulum Retention and Protein Body Formation.

Abstract

y-Zein is a maize storage protein synthesized by endosperm cells and stored together with a-and P-zeins in specialized organelles called protein bodies. Previous studies have shown that in maize there is only one type of protein body and it is derived directly from the endoplasmic reticulum (ER). In this article, we describe the domains of y-zein involved in ER retention and the domains involved in protein body formation. To identify the signal responsible for y-zein retention in ER-derived protein bodies, DNAs encoding various deletion mutants of y-zein were constructed and introduced into Arabidopsis as a heterologous system. By using pulse-chase experiments and immunoelectron microscopy, we demonstrated that the deletion of a proline-rich domain at the N terminus of y-zein puts an end to its retention in the ER; this resulted in the secretion of the mutated protein. The amino acid sequence of y-zein necessary for ER retention is the repeat domain composed of eight units of the hexapeptide PPPVHL. In addition, we obsenred that only those y-zein mutants that contained both the proline-rich repeat domain and the C-terminal cysteine-rich domain were able to form ER-derived protein bodies. We suggest that the retention of y-zein in the ER could be a result of a protein-protein association or a transient interaction of the repeat domain with ER membranes.

Coexpression of the maize delta-zein and beta-zein genes results in stable accumulation of delta-zein in endoplasmic reticulum-derived protein bodies formed by beta-zein.

“…The 8-zein protein bodies, similar to those of p-zein and -/-zein, appear to be retained within the ER. The 5-zein protein bodies, however, are morphologically distinct from the protein bodies formed by p-zein (Bagga et al, 1995) or -y-zein (Geli et al, 1994) or those formed in maize endosperm (Lending et al, 1988), suggesting that each type of zein has intrinsic structural properties that influence the morphology of the protein body. Moreover, the results presented here demonstrate that vegetative tissues of a dicot plant can be induced to produce protein bodies that are typical of seeds of cereal plants.…”

“…We have demonstrated that 8-zein, like p-zein (Bagga et al, 1995) and -/-zein (Geli et al, 1994), is capable of forming protein bodies by itself. The 8-zein protein bodies, similar to those of p-zein and -/-zein, appear to be retained within the ER.…”

“…It has been proposed that y-zein or p-zein or both are necessary for the assembly of the protein bodies (Lending and Larkins, 1989). Some studies suggest that an ER transmembrane domain exists in -y-zein that acts as a nucleating factor in protein body formation (Torrent et al, 1994), but a recent report refuted the presence of a transmembrane region in the -y-zein protein (Lee et al, 1995). Little is known about the interactions among the different zeins and how the interactions contribute to the formation of protein bodies.…”

“…In the case of -y-zein, Geli et al (1994) determined that a proline-rich region composed of repeats of a hexapeptide (PPPVHL) in the N-terminal domain is responsible for ER retention, whereas both the proline-rich repeat and the C-terminal cysteine-rich domains are needed for protein body biogenesis. p-Zein, which can form protein bodies alone, does not have these proline-rich repeats, but it does contain a short cysteine-rich domain (Shewry et al, 1995).…”

“…The -y-zein and p-zein coding sequences have been expressed individually in transgenic Arabidopsis (-y-zein) and tobacco 0-zein) plants by using the cauliflower mosaic virus (CaMV) 35S promoter, and in both cases, the proteins were retained in ER-derived protein bodies (Geli et al, 1994;Bagga et al, 1995). In the case of -y-zein, Geli et al (1994) determined that a proline-rich region composed of repeats of a hexapeptide (PPPVHL) in the N-terminal domain is responsible for ER retention, whereas both the proline-rich repeat and the C-terminal cysteine-rich domains are needed for protein body biogenesis.…”