In Vitro Synthesis of a Precursor to the Methionine-rich Polypeptide of the Zein Fraction of Corn

Abstract

The messenger ribonucleic acid fraction isolated from a protein bodyenriched fraction of developing corn (Zea mays L.) endosperm stimulated the incorporation of radioactive amino acids into at least five polypeptides when added to a wheat germ extract capable of protein synthesis. Of these, the two major polypeptides formed with messenger from freshly frozen corn were identified as precursors to zein A and B, the two major polypeptides of the prolamine fraction of corn meal (21,600 and 19,600 molecular weight). The identification was based on the relative incorporations of radioactive leucine, lysine, and methionine, and the susceptibility of the zein A precursor, but not the zein B precursor to cleavage wtih cyanogen bromide. Using extracts from stored frozen corn, a third polypeptide of 14,500 molecular weight was identified as a major in vitro product. It was preferentially labeled with methionine and slightly larger than a similar peptide in the zein fraction of corn meal. Two other polypeptides of still lower molecular weight could be detected above the background of probably incomplete polypeptides.A major portion of the storage protein of corn endosperm is synthesized on ribosomes bound to ER and protein body membranes (4). Protein bodies are accretions of insoluble protein surrounded by a membrane on which ribosomes are bound. The bodies arise by pinching off from the ER (6). The polypeptide products of translation of these ribosomes must pass through the membrane and accumulate in the interior of the protein body. In other cases where a newly synthesized polypeptide is transported through a membrane, the initial product of translation has an Nterminal extension rich in hydrophobic amino acids, which it is postulated serves as a signal for transmembrane movement of the polypeptide (2). The bound ribosomes of corn have provided the starting material for the isolation of mRNA specific for the synthesis of corn storage proteins (5,9).The protein inside the protein bodies is of the zein or alcoholsoluble fraction of corn proteins. Two polypeptides, zein A and zein B (21,600 and 19,600 mol wt, respectively) make up the bulk of this protein fraction. These correspond to Z1 and Z2 of Lee et al. (11). Several other polypeptides of lower mol wt are also present in zein extracts (11). Extraction of corn meal with an alcoholic solvent followed by an alcoholic solvent containing a reducing agent has led to the isolation of two corn prolamine fracti'ons, designated zein I and zein II. Zein II contains onefourth to one-half the amount of protein in zein I (7,21 I protein with 10 to 20o of a protein fraction rich in methionine and glycine and deficient in leucine, isoleucine, phenylalanine and aspartic acid and/or its amide (7,21). This hypothesis has been confirmed by electrophoresis in urea gels (19,21), gel filtration in the presence of urea (19), and 20). The zein II fraction contains all of the polypeptides of the zein I fraction, but in addition contains two or more polypeptides of greater cathodal mobi...

In maize, glutelin-2 and low molecular weight zeins are synthesized by membrane-bound polyribosomes and translocated into microsomal membranes

“…In maize, the intracellular site of synthesis and cotranslational modifications of polypeptides corresponding to zeins-1 (19, 21.5, 22 kD Z1) have been extensively studied ( 3 -6, 14-16). However, the picture for the biosynthesis of low molecular weight zeins (10,14,15 kD Z2) is much less complete (22,30).…”

In vivo synthesis and processing of cereal lectins

“…Recent work on the synthesis of plant proteins has shown that the primary translation products of several classes of plant proteins are proteolytically cleaved during translation whereby a signal peptide is removed and the protein transported through the membrane of the endoplasmic reticulum. Among them are storage proteins as pea, soybean and broad bean vicilins [13,32,35], barley prolamins [7] and zein [5,16,19]. Other examples are enzymes such as c~-amylase in wheat [4], barley [23] and rice [20] and a protease inhibitor from tomato leaves [24].…”

Precursor Forms of Pea Vicilin Subunits

“…The reasons for these differences are not understood at present. Evidence has been obtained for similar precursors in the storage proteins of barley and maize (6,8,18,24,36) and Burr et al (8) presented preliminary evidence that the additional sequence was located at the amino-terminus of a zein polypeptide. The storage proteins of French bean have also been shown to be synthesized in vitro as polypeptides of different size to those found in vivo (15), but it is not known whether these precursors are affected by membranes.…”

“…The biosynthesis of storage proteins of legumes and cereals has been studied during seed maturation by in vivo pulse labeling (5,34) and by cell-free translation of polyribosomes and RNA (3,5,6,8,9,13,15,16,18,22,24,29,35,36). The synthesis and accumulation of individual storage proteins and their component polypeptides occur at well-defined stages during seed development (for review, see ref.…”

“…These include glycosylation (2,10,12) and post-translational cleavage of a high mol wt precursor (9,33) as well as other undefined changes which result in altered electrophoretic mobility of polypeptides (34) and proteins (14,26,28). There is also evidence which suggests that some of the newly synthesized storage protein polypeptides of cereals (6,8,18,24,36) and legumes (15,16) are synthesized as short-lived precursors which may undergo modification during or after translation. In the case of the cereals, the precursors appear to be equivalent to the secretory and membrane proteins of mammalian cells in that a "signal" peptide may occur on the polypeptide.…”