Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6448-6452, December 1979 Cell Biology Synthesis and processing of maize storage proteins in Xenopus laevis oocytes (signal peptide/seed protein/zein mRNA) BRIAN A. LARKINS*, KARL PEDERSEN*, AVTAR K. HANDA*, WILLIAM J. HURKMAN*, AND L. D. SMITHt *Department of Botany and Plant Pathology, and tDepartment of Biology, Purdue University, West Lafayette, Indiana 47907 Communicated by Myron Braklce, September 17, 1979 ABSTRACT Xenopus oocytes injected with zein mRNAs identified. In this communication we report experiments to efficiently synthesized maize storage proteins for prolonged determine whether maize seed storage proteins can be syn- periods. Under optimal conditions, zein was synthesized at 3 thesized in Xenopus oocytes and whether the signal peptides ng/hr and represented approximately 10% of the total protein synthesized in the oocyte. The mRNA from the normal maize associated with them are processed. inbred line directed synthesis of all the major zein components; however, products of mRNA from the opaque-2 mutant did not MATERIALS AND METHODS contain the largest zein component. Zein proteins synthesized Preparation of mRNA. Zein mRNA was isolated from frozen in the oocyte were 2000 daltons smaller than proteins synthe- kernels of the maize inbred line W64A and its opaque-2 mutant sized by cell-free translation of mRNAs in the wheat germ and reticulocyte systems. This result, which suggested that the oocyte as described (5). Polyadenylylated RNA from membrane- processed prezein polypeptides into native zein proteins, was bound polysomes of kernels 22 days after fertilization was confirmed by amino-terminal sequence analysis of zein proteins fractionated on linear logarithmic sucrose gradients to remove from the oocytes. Cyanogen bromide cleavage of translation contaminating 18S rRNA, and RNA sedimenting at 13 S was products from oocytes and the wheat germ system confirmed precipitated with 2 vol of absolute ethanol. The RNA was later the existence of several proteins within each of the major zein pelleted by centrifugation and dissolved in sterile H20. components. However, we were unable to detect the presence Preparation and Injection of Oocytes. X. laevus were ob- of signal sequences on zein peptides by this technique. tained from South Africa and maintained as described (15). Zein, the major storage protein fraction in maize (Zea nays L.) Ovaries were removed from frogs anesthesized by hypothermia, seed, consists of a group of ethanol-soluble proteins that are and oocytes were defolliculated manually in sterile Ringer's localized in protein bodies. Sodium dodecyl sulfate (Na- solution. Stage-six oocytes were transferred to Eppig and Du- DodSO4)/polyacrylamide gel analysis of zein proteins reveals mont's medium (16) and, after "equilibration" for 1-2 hr. were two major components of Mr 22,000 and 19,000 and small microinjected individually with 20 nl of mRNA dissolved in amounts of 15,000 and 10,000 Mr proteins (1, 2). When these sterile H20 or Ringer's solution. After an additional 24 hr of proteins were analyzed by isoelectric focusing between pH 5 incubation, the mRNA-injected oocytes were injected with 20 and 9, as many as 28 different species were resolved (3), indi- nl (0.4 ,uCi; 1 Ci = 3.7 X 1010 becquerels) of [3H]leucine (Am- cating charge heterogeneity among zein polypeptides. A ersham) or [3H]leucine plus ['4C]isoleucine (0.04 ,uCi, Amer- structural basis for at least part of this heterogeneity was sham). The label was prepared by addition of 0.25 ,umol of demonstrated by amino-terminal sequence analysis of the major carrier leucine or isoleucine to 1 ml of [3H]leucine (1 mCi/ml). zein components from the inbred line W64A (4), where each After evaporation to dryness in a desiccator, the sample was of the major components gave a minimum of two different dissolved in 50 ,l of sterile water. amino-terminal sequences. Measurement of Protein Synthesis and Amino Acid Pool During development of the maize seed, zein proteins are Size. Incorporation of [3H]leucine into total protein was de- synthesized in the endosperm, where they form protein bodies termined by placing two oocytes in hot (600C) 0.5 M perchloric within the rough endoplasmic reticulum (RER) (5). mRNAs acid for 30 min, followed by a 30-min rinse with distilled H20 that direct the synthesis of zein proteins have been isolated from (17). .The acid-extracted oocytes were then dissolved in NCS polysomes of the RER, and in the wheat germ cell-free system solubilizer (New England Nuclear) and radioactivity was de- these mRNAs direct the synthesis of proteins that are 2000 termined in a Beckman LS 230 liquid scintillation counter after daltons larger than native zein proteins (5-7). However, when addition of 10 ml of Omnifluor (New England Nuclear). For intact RER vesicles are placed in the wheat germ system, pro- measurement of incorporation into zein proteins, two oocytes teins of the same molecular weight as native zein proteins are were homogenized in 0.25 ml of 70% ethanol containing 1% synthesized (5). These observations suggest that these plant 2-mercaptoethanol. After extraction at 60°C for 10 min, in- storage proteins, like animal secretory proteins, contain signal soluble proteins were precipitated by centrifugation in a Sor- peptides that direct their synthesis into the RER (8). vall-SS 34 rotor at 6000 rpm for 10 min. Aliquots of the etha- Xenopus laevis oocytes efficiently translate heterologous nol-soluble proteins were spotted on Whatman 3MM filter mRNAs (9-11) and can process signal sequences when mRNAs papers and hot trichloroacetic acid-insoluble radioactivity was from animal cells that direct the synthesis of preproteins are determined (18). microinjected (12-14). To our knowledge similar studies with The endogenous pool of leucine in oocytes incubated for 24 plant preprotein mRNAs have not been reported nor, for that hr in Eppig and Dumont's medium (16) was determined by matter, have any plant proteins with signal peptides been analyzing the cold 30% trichloroacetic acid-soluble extract of 400 manually defolliculated oocytes. After the acid was re- the was and The publication costs of this article were defrayed in part by page moved by ether extraction, homogenate lyophilized charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: NaDodSO4, sodium dodecyl sulfate; RER, rough en- this fact. doplasmic reticulum. 6448 Downloaded by guest on September 26, 2021 Cell Biology: Larkins et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6449 analyzed on a Beckman model 120C amino acid analyzer. 1 2 3 4 5 6 7 8 Based on the average of four separate determinations, the en- 12S XO WG 125 X o WG XO XO dogenous leucine pool was determined to be 160 25 pmol/ oocyte. Rates of total protein and zein synthesis were determined by the method of Shih et al. (17), assuming an endogenous leucine pool of 160 pmol/oocyte and an injected leucine concentration of 100 pmol/oocyte. Rates of synthesis were calculated as- suming a 20% leucine content for the maize zein polypeptides 22,000_ (1, 2) and 10% leucine for the endogenous oocyte proteins. No 19.000- tA correction was made for zein content among the total oocyte proteins. 1 5,000 In Vitro Protein Synthesis. A standard cell-free protein- synthesizing system (19) was prepared from wheat embryos as Si& described (5). After translation of zein mRNA, the reaction mixture was brought to 70% with absolute ethanol and held at FIG. 1. NaDodSO4/polyacrylamide gel analysis of 70% ethanol- 60'C for 10 min. Ethanol-insoluble proteins were removed by soluble proteins synthesized in Xenopus oocytes. 125I-Labeled native zein proteins as well as normal and opaque-2 mRNA translation centrifugation at 6000 rpm for 10 min. An mRNA-dependent products from a wheat germ cell-free system were analyzed for com- cell-free translational system was also prepared from a rabbit parison. Although the tyrosine content ofthe different zein proteins reticulocyte lysate by micrococcal nuclease digestion (20). After is similar (1, 2), the 15,000 Mr protein was preferentially labeled by a 60-min translation, zein proteins were extracted by adding 125I. Samples 1 and 4, 25I-labeled normal zein, 190,000 cpm; samples absolute ethanol to a final concentration of 70%, and insoluble 2, 5, and 8, [3H]leucine-labeled ethanol-soluble proteins from oocytes proteins were removed by centrifugation. Radioactive protein injected with normal zein mRNA, 50,000 cpm; samples 3 and 6, [3H]leucine-labeled products of opaque-2 and normal zein mRNA samples were lyophilized and analyzed on 12.5% Na- translation, respectively, from a wheat germ cell-free system, 30,000 DodSO4/polyacrylamide gels (acrylamide/bisacrylamide, 75:1) cpm; sample 7, [3H]leucine-labeled ethanol-soluble proteins from as described (5). Proteins cleaved with cyanogen bromide were Xenopus oocytes without injected mRNA, number of oocytes analyzed on 25% NaDodSO4/polyacrylamide gels (acrylam- equivalent to samples 2, 5, and 8. ide/bisacrylamide, 150:1). Radioactivity was detected by fluorography with pre-exposed Kodak RP X-Omat film (21). shown in Fig. 2, injection of [3H]leucine at relatively low spe- Native zein proteins were labeled with 125I by the chloramine-T cific activity (4 Ci/mol) expanded the endogenous pool suffi- method (22). ciently to generate linear incorporation into both total protein Amino-Terminal Sequence Analysis of Zein Proteins. Zein and zein proteins for at least 90 min. This allowed accurate proteins synthesized in oocytes or the wheat germ system were estimation of the absolute rates of protein synthesis as described double-labeled with [3H]leucine and [14C]isoleucine and mixed (17). In this particular experiment, the oocytes were injected with small amounts of dansylated native zein proteins.