Transfer of in vitro synthesized barley endosperm proteins into the lumen of the endoplasmic reticulum

Abstract

Microsomes were prepared from 20 day old Bomi barley endosperm by sucrose density gradient centrifugation. Electron microscopy revealed the presence of ribosome-studded vesicles in the material banding at the 1.75-2.26 M sucrose interface. The isolated microsomes were active in the wheat-germ cell-free protein synthesizing system. Hordeins were present among the in vitro synthesized products and were identified by their solubility in 55% isopropanol and by their co-migration with native hordeins on SDS-polyacrylamide gels. The microsomeand polysome-directed, in vitro synthesized hordeins were analysed before and after chymotrypsin treatment by SDS-polyacrylamide gel electrophoresis. The hordeins were degraded when polysomes were used as a template. However, hordeins synthesized on microsomes showed significant protection from proteolysis. This protection could be abolished by treatment with membrane-solubilizing detergents. The reported experiments show that hordeins synthesized on microsomes were discharged vectorially into the lumen of the microsomes.

In vitro synthesis and transport of barley endosperm proteins: Reconstitution of functional rough microsomes from polyribosomes and stripped microsomes

“…Rough microsome fractions, removed from discontinuous sucrose gradients (8), were diluted with an equal volume of HKM buffer B, and fixed with 2% g[utaraldehyde for lh at 0~ The fixed material was pelleted in conical tubes with a bench centrifuge, and then resuspended in 60mM-phosphate buffer pH 7.3, containing 2% glutaraldehyde. After further centrifugation, the pellets were given three 10 min washes in 60mM-phosphate buffer pH 7.3, and then postfixed with 1% osmium tetroxide in the same buffer.…”

“…Cell-free protein synthesis was performed as previously described (8), except that the incubation was for 30 rain.…”

“…Rough microsomes, prepared from homogenates of Bomi and mutant endosperm, were isolated from discontinuous sucrose gradients in which they banded at the interfaces of 1.5-1.7M (band II) and 1.7-2.26M (band 1), (8). Polysomes from Bomi and mutant endosperms were obtained by Triton-extraction of the membrane pellet (5).…”

“…During endosperm development in barley grains, some 20 days after anthesis, a large part of the protein-synthesizing machinery is directed towards the synthesis of storage proteins (4). Among the major storage proteins, the hordeins are synthesized in vitro on membrane-bound polysomes (5) and are discharged vectorially into the lumen of the endoplasmic reticulum (8). The primary translation products of purified hordein mRNA's are 2 kilodaltons larger than the native hordein polypeptides (6), while the hordein polypeptides synthesized by rough microsomes are close in size to the native hordein polypeptides (8).…”

“…Among the major storage proteins, the hordeins are synthesized in vitro on membrane-bound polysomes (5) and are discharged vectorially into the lumen of the endoplasmic reticulum (8). The primary translation products of purified hordein mRNA's are 2 kilodaltons larger than the native hordein polypeptides (6), while the hordein polypeptides synthesized by rough microsomes are close in size to the native hordein polypeptides (8). Thus, there is strong evidence that the synthesis and deposition of barley endosperm storage proteins follows the scheme outlined for animal secretory proteins in the ,,signal hypothesis,, (1,2).…”