Department of Biochemistry, College of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan
Abbreviations: CBB-Coomassie Brilliant Blue; DTT-dithiothreitol; HEPES-N-2-
hydroxyethylpiperazine-N'-2ethanesulfonic acid; PAGE-polyacrylamide gel
electrophoresis; PB-protein body(ies); PVS-poly vinyl sulfate; SDG-sucrose
density gradient; SDS-sodium dodecylsulfate.
Storage proteins of rice seeds are accumulated into two different types of protein bodies (PB). PB-I and PB-II accumulate prolamin and glutelin as their major components, respectively (Tanaka et al. 1980). PB-I is hard to be digested and the components have low nutritive value (Tanaka et al. 1975). The content of this type exceeds more than 30% of total endosperm protein in some Indica varieties (Ogawa et al. 1987). For this reason, improvement of the digestibility and nutritive value of PB-I is one of important problems to be solved in the future.
In an attempt to solve this problem, we introduced maize polypeptides into rice PB-I. Maize polypeptides could be replaced with a polypeptide which is easily digested and possesses a high nutritive value, such as bovine casein. We use maize PB as one of rice PB-1 type protein bodies. Probably, sorghum and pearl millet protein bodies also belong to this type.
The origin of rice PB-I is the endoplasmic reticulum membrane and pictures very similar to maize PB were obtained when developing PB were observed using a transmission electron microscope (Burr and Burr 1976; Yamagata and Tanaka 1986a). PB-II, however, appears to be derived from the vacuole membrane, although all rice storage polypeptides are synthesized on membrane-bound polysomes (Yamagata and Tanaka 1986a).
Our recent electron microscopic and biochemical work suggests that there must be at least two different types of membranes in a rice endosperm cell. One develops into PB-I and the other into PB-II via Golgi machinery (Yamagata and Tanaka 1986a).
We recently constructed a cDNA library from developing rice endosperm mRNAs and isolated clones which code for the rice 10 kDa prolamin. Structural analysis of the entire cDNA of this prolamin revealed that the signal-sequence composed of 24 amino acids had quite a high homology to that of major prolamins of maize (Masumura et al. 1988).
Similar features of maize PB and rice PB-I, and the homology in the signal- sequence region of the rice 10 kDa prolamin precursor with zein precursors imply that the same or very similar mechanisms are operating for rice PB-I and maize PB formation. Especially, common amino acid sequences distributed in the signal-sequence region of the rice 10 kDa prolamin precursor and zein precursors are supposed to be important for the recognition of PB-I type membranes.
Rice plants (Nipponbare) were grown at monthly intervals in a greenhouse at a temperature range of 25-30 degrees C. Maize plants, variety Honey Bantum, were grown under a similar condition as for rice. Messanger RNA was isolated from the developing rice and maize seeds by phenol extraction followed by oligo(dT) cellulose column chromatography as previously described (Yamagata et al. 1986b).
From developing rice seeds (15 days after flowering) or maize seeds (30 days after flowering), endosperm was squeezed into an Eppendorf tube (1.5 ml) and homogenized using a glass rod with a buffer of 20 mM HEPES, 2 mM DTT, 250 mM sucrose, 5 mM MgCl\2\, 0.001% PVS at pH 7.6 at 0 degrees C. The material was centrifuged at 100 X g for 1 min to remove most of the starch granules, and then the supernatant was further centrifuged at 2,000 X g for 2 min. The resulting precipitate was collected and used for experiments requiring the PB fraction.
Translation of the poly(A)+RNA fraction from rice endosperm or maize endosperm was carried out in a wheat germ cell-free system (Amersham) using (3H)-leucine. About 1 ug of total maize poly (A)+RNA was translated in a wheat germ cell-free system which included about 36 uU of rice PB fraction (OD\500\=1.0: 1U/ml) obtained from 10 DAF seeds. The PB were recovered by centrifugation at 2,000 X g for 2 min. Polypeptides associated with the recovered PB fraction were fractionated using a SDS-PAGE (Laemmli 1970) and fluorographed (Bonner and Laskey 1974). To examine the status of the PB associated polypeptides, proteinase K treatment with or without detergent was carried out.
As shown in Fig. 1, maize 19 kDa prolamin was preferentially accumulated in rice PB. Many maize polypeptides were associated with rice PB, and some of them were stably retained in PB even after the PB fraction was treated with proteinase K. This result suggests that many polypeptides synthesized in the developing maize endosperm are able to be associated with the rice endosperm PB and some polypeptides are able to travel through the rice PB membrane.
Fig. 1. Organization of mazin zein polypeptides into rice protein bodies.
1 ug of maize poly(A)+RNA fraction was translated in a wheat germ cell-free system with 36 uU of rice PB fraction in a total volume of 12 u1. After incubation for one hour at 25 degrees C, the reaction mixtures were treated with 0.2 mg/ml proteinase K for 45 min at 0 degrees C with or without 1% SDS. Then the reaction mixture was centrifuged at 2,000 X g for 2 min, and the precipitated material was fractionated by SDS-PAGE, and fluorographed. T: Total translation product. PB: Rice PB recovered. S: Soluble fraction of the reaction mixture.
The zein polypeptides residing within the rice PB membrane became digestible by
proteinase K when the PB membrane was removed by the SDS treatment.
Polypeptides resistant to proteinase K treatment were appreciably decreased
when in vitro translation was carried out without rice PB.
These results indicate that maize endosperm polypeptides were able to target the surface of rice PB and some of them were stably accumulated within the surface membrane of rice PB.
Fig. 2 and Fig. 3, are experiments complementary to that shown in Fig. 1. In these experiments total rice poly(A)+RNA obtained from 10 DAF developing seeds was translated in a wheat germ cell-free system in the presence of the PB fraction of developing maize seeds. As shown in Fig. 2, the reaction mixture was fractionated by a SDG to recover maize PB.
Judging from the CBB staining shown in panel B, maize PB were recovered mainly in fraction 4. Result in panel A shows that among many nascent polypeptides directed by total mRNA of developing rice seeds, only a limited number of polypeptides were associated with maize PB. Among them. sorting of prolamins is most remarkable and association with maize PB is clear in the comparison of results in comparing panels, A and B.
In Fig. 3, fluorograms obtained after various treatments were compared. In lane TP and PB, different major bands were observed as indicated by stars on lane PB, and open circles on lane TP. The bands marked with stars are exactly the same size as those observed in mature rice endosperm. This result means that the rice prolamin precursors synthesized in the wheat germ cell-free systme were sorted, traveled through the maize PB membrane and were then processed into mature rice prolamins. Rice glutelin was also targeted to maize PB as indicated by an arrowhead near the top of lane PB. However, this band completely disappeared following the pepsin treatment, although processed rice prolamin polypeptides (star marks) showed resistance.
Our recent finding about the cDNA structure of 10 kDa prolamin (lambdaRP10) showed that the signal amino acid sequence has extremely high homology to that of maize prolamin (lambdaZG99; Pedersen et al. 1982), although no remarkable homologous sequences were observed within the prolamin structural region. As shown in Figs. 2 and 3, rice prolamins were completely processed to form mature prolamin polypeptides during deposition into the maize PB. Similarly, maize prolamin polypeptides successfully sorted into rice PB (Fig. 1).
From the results, we conclude that signal-sequence with a portion of the N- terminal region is required for the accurate sorting of a prolamin polypeptide by the PB-I type membrane.
We continue this study to elucidate the mechanism operating during polypeptide acumulation into the PB-I type protein bodies. This work has been supported by grants from the Ministry of Education, Science and Culture (Japan), the Ministry of Agriculture, Forestry and Fisheries (Japan) and the Rockefeller Foundation.
Fig. 2. Specific sorting of rice prolamin polypeptides into maize
protein bodies.
5 ug of rice poly(A)+RNA fraction was translated in a wheat germ cell-free system with about 180 uU of maize protein body fraction in a total volume of 50 ul. After incubation for one hour at 25 degrees C, the reaction mixture was layered on a 50%-70% sucrose density gradient in a Beckman TLS55 polyallomer tube and centrifuged at 60,000 X g for 25 min. The fractions were collected after piercing the bottom of the tube. Maize PB was recovered mainly in fraction 4 when the pelleted material of each fraction was fractionated by SDS- PAGE, then stained by CBB (Panel B). Panel A, a fluorogram obtained after each SDG fraction was fractionated by SDS-PAGE. Panel C, the percentage of sucrose corresponding to the different fraction.
The fluorogram shown in the right-hand margin of panel A is the total translation product directed by the total poly(A)+RNA fraction obtained from developing rice endosperm.
Fig. 3. Processing of rice prolamin polypeptides inside of maize protein
bodies.
The rice poly(A)+RNA fraction was translated in the presence of maize PB in a wheat germ cell-free system. Ttranslational product was analyzed by fluorography after fractionation by SDS-PAGE. TP: Total translation product directed by poly(A)+RNA of rice endosperm without addition of maize protein bodies. PB: Maize PB fraction recovered after incubation and centrifugation. PEP: Maize protein body fraction recovered after pepsin treatment was added to the PB franction in the center lane. (open circle): Precursor form corresponding to rice prolamin polypeptide. (Star): Processed (mature) rice prolamin polypeptide.
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