Shin-ichiro Ejiri

Moonlighting Functions of Polypeptide Elongation Factor 1: From Actin Bundling to Zinc Finger Protein R1-Associated Nuclear Localization

Eukaryotic polypeptide elongation factor EF-1 is not only a major translational factor, but also one of the most important multifunctional (moonlighting) proteins. EF-1 consists of four different subunits collectively termed EF-1αββ′γ and EF-1αβγδ in plants and animals, respectively. EF-1α•GTP catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome. EF-1ββ′γ (EF-1β and EF-1β′), catalyzes GDP/GTP exchange on EF-1α•GDP to regenerate EF-1α•GTP. EF-1γ has recently been shown to have glutathione S-transferase activity. EF-2 catalyzes the translocation of peptidyl-tRNA from the A-site to the P-site on the ribosome. Recently, molecular mimicry among tRNA, elongation factors, releasing factor (RF), and ribosome recycling factor (RRF) has been demonstrated and greatly improved our understanding of the mechanism of translation. Moreover, eukaryotic elongation factors have been shown to be concerned or likely to be concerned in various important cellular processes or serious diseases, including translational control, signal transduction, cytoskeletal organization, apoptosis, adult atopic dermatitis, oncogenic transformation, nutrition, and nuclear processes such as RNA synthesis and mitosis. This article aims to overview the recent advances in protein biosynthesis, concentrating on the moonlighting functions of EF-1.

Molecular characterization of rice genes specifically expressed in the anther tapetum

In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5′-upstream region was found to regulate β-glucuronidase expression in the tapetum of transgenic tobacco. A set of 5′ deletions was also generated and a 1095 bp 5′ region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco.

The purification of methionine sulfoxide reductase from Escherichia coli

A sensitive assay, based on the acylation of tRNAMet, has been developed to measure the enzymatic reduction of methionine sulfoxide to methionine. Using this assay, methionine sulfoxide reductase has been purified to near homogeneity from extracts of Escherichia coli.

ISOLATION, CHARACTERIZATION AND MRNA EXPRESSION OF FOUR CDNAS ENCODING TRANSLATION ELONGATION FACTOR 1A FROM RICE (ORYZA SATIVA L.)

Four different cDNA clones encoding protein synthesis elongation factor 1A, eEF1A, were isolated from rice (Oryza sativa L.). The genes encoded by these cDNAs were designated rice elongation factor 1A genes refa1, refa2, refa3 and refa4. The genes encoded identical eEF-1A polypeptides and shared high amino acid identity with eEF1A of other eukaryotes. Southern blot analysis suggested that some of these refa genes may be organized in a cluster on the same chromosome within a short distance. PCR analysis of rice genomic DNA showed that refa1 and refa4, and refa3 and refa2 are in neighboring locations on the rice genome. The mRNAs of the four refa genes accumulated to nearly equal levels in a variety of tissues and at different stages of growth. Suspension-cultured cells were the most abundant in refa mRNAs. Dormant seeds contained a small amount of the four refa mRNAs. Transcript accumulation was highly induced after seed germination, and the same expression levels were maintained even in old leaf blades of mature plants.

Isolation of genes abundantly expressed in rice anthers at the microspore stage

A cDNA library of rice (Oryza sativa L.) has been constructed from anthers at an early stage of pollen development. By differential screening of the library, we have isolated cDNAs of two genes, designated as Osc4 and Osc6, that are abundantly expressed in anthers containing tetrads and uninucleate microspores, but are not expressed in leaves or roots. Expression of Osc4 is absent in mature anthers, while Osc6 is present although the expression decays during pollen maturation. A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown significant homology to known molecules.

Elongation factor 1 from the silk gland of silkworm Purification and some properties of its γ subunit having EF-1b activity

Elongation factor 1 (EF-l), the factor responsible for binding aminoacyl-tRNA to ribosomes, exists in multiple forms in a variety of different eukaryotes [ 1,2] . The heavier forms (EF-1, , mol. wt > 150 000) represent aggregates of a lighter form(EF-lL, mol. wt approx. 60 000) [3--S]. On the other hand, we previously noted that silk gland EF-l(APase I) consisted of three different subunits ((Y, fl and y) [6] . Their molecular weights were estimated to be about 63 000,60 000 and 30 000, respectively. Furthermore EF-I, was resolved into two complementary factors, EF-la(APase I) and EF-lb(APase II), which were thought to correspond to prokaryotic EF-Tu and EF-Ts, respectively [7]. Similar factors were also observed in pig liver [8] , rabbit reticulocyte [9] , wheat embryo [lo] and Alfemiu salina cysts [ 1 l] . From these results it is of interest and important to compare the properties of each subunit in detail. However, for this purpose the recovery of each subunit of silk gland EF-1 was low in the former procedure [7] because only a part of it was dissociated under the undenaturing conditions. Thus, we tried to dissociate and purify each subunit under denaturing conditions. In this paper we describe the procedures for the purification and some properties of the 7 subunit which showed EF-lb(EF-TSUke) activities.

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 α-type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.

The promoter from the rice nuclear gene encoding chloroplast aldolase confers mesophyll-specific and light-regulated expression in transgenic tobacco

The rice genome contains at least four separate loci that encode aldolase isozymes. Among these, the aldolase P (AldP) gene, a nuclear gene coding for chloroplast aldolase, is expressed predominantly in the leaf blade mesophyll cells in rice. To dissect promoter elements that regulate such tissue- or cell type-specific expression, we constructed variousAldP promoter-β-glucuronidase (GUS) fusion genes and transferred them intoNicotiana tabacum (tobacco) plants. Analysis of GUS activities in the transgenic tobacco revealed the presence of at least two elements within 2.0 kbAldP promoter region. One is located within the segment from position − 2.0 kb to − 1.2 kb and acts as a negative element. The other is a positive element located between − 1.2 kb and − 0.31 kb that confers developmentally regulated, mesophyll cellspecific expression. In addition, the 1.2 kb rice promoter segment flanking the transcription start site contains an element(s) that serves as target for light induction in tobacco. The results suggest that theAIdP gene promoter of rice, a monocot promoter, can function in an essentially physiological manner in the dicot tobacco plant.

A factor protecting mammalian [75Se]SeCys-tRNA is different from EF-1α

In Escherichia coli, an elongation factor (EF‐Tu‐like) specific to SeCys‐tRNA, SELB, has been identified; however, a mammalian counterpart of SELB has not been reported to date. We searched for and found this factor in bovine liver extracts using the assay of [75Se]SeCys‐tRNA protecting activity against alkaline hydrolysis (SePF activity). We found SePF activity in the protein extracts of the precipitate (microsomal fraction) collected at 150,000 × g from bovine liver. The proteins were separated by Sephacryl S‐300 chromatography, and the SePF and EF‐1α activities were found in the same fraction, indicating that SePF and EF‐1α have the same molecular mass (approximately 50 kDa). We then chromatographed this active fraction using CM‐Sephadex C‐25 columns. The SePF activity was eluted after the peak of EF‐1α activity. This result indicated that this SePF activity was not dependent on EF‐1α. In addition to performing these two chromatographies, we investigated pure EF‐1α from Bombyx mori but could not detect any SePF activity in B. mori EF‐1α. Thus we showed that the SePF activity in bovine liver differs from that of EF‐1α in eukaryotes. Therefore the factor protecting [75Se]SeCys‐tRNA in bovine liver is not EF‐1α and must be a SELB‐like factor.

Genomic structure of the rice aldolase isozyme C-1 gene and its regulation through a Ca2+-mediated protein kinase-phosphatase pathway

Complementary and genomic DNA clones coding for aldolase C-1, the fourth-type isozyme of aldolase in rice Oryza sativa L., have been characterized. The organization of the gene is quite similar to those encoding rice aldolase C-a and a maize cytoplasmic-type aldolase, in that introns are located in the same position. Amino acid sequences are highly conserved among cytoplasmic aldolases in plants. Expression of the gene in rice callus is activated by a protein phosphatase inhibitor okadaic acid, and is inhibited in the presence of thapsigargin, a reagent which increases calcium influx into the cytoplasm. The inhibition is rescued by the simultaneous addition of protein kinase inhibitor H-7. Thus, it is suggested that expression of the aldolase C-1 gene is regulated through a signal transduction pathway involving a Ca2+-mediated protein kinase-protein phosphatase system.