u lar Compartmentalization of Maize Storage Proteins in Xenopus Oocytes Injected with Zein Messenger RNAs 292

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1 .10-1 .12 g/cm 3), mitochondria (densities = 1 .14 and 1 .16 g/cm 3), yolk platelets (density = 1 .21 g/cm 3), and a dense matrix material (density = 1 .22 g/cm) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of-1 .22 g/cm 3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypep-tides. The principal storage protein fraction in maize endosperm consists of a group of alcohol-soluble proteins called zein. During endosperm development, zein proteins are synthesized and deposited as protein bodies within the rough endosplasmic reticulum (1, 2). An analysis of zein proteins by SDS-polyacryl-amide gel electrophoresis reveals two major components with 19,000 and 22,000 mot wt, as well as several minor components of 10,000 and 15,000 mot wt (3). However, isoelectric focusing of zein proteins resolves up to 28 polypeptides (4), indicating substantial charge heterogeneity among them. Zein proteins are synthesized by membrane-bound polyri-bosomes (5), and when these polysomes or purified zein mRNAs are translated in vitro, the proteins synthesized are 2,000 mot wt larger than native zein polypeptides (2, 6, 7). In a previous study, we reported that when zein mRNAs were injected into Xenopus oocytes, zein proteins were synthesized with molecular weights identical to those of the native poly-peptides (8). We also demonstrated that proteins from both sources had identical amino terminal sequences. To determine whether the synthesis and processing of zein polypeptides in oocytes are accompanied by protein body formation, we have examined the subcellular distribution of zein proteins. We considered it important for …