Masazumi Miyazaki

Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae

Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine, a modification of a specific lysine residue in the precursor of eukaryotic translation initiation factor 5A. We have purified deoxyhypusine synthase from yeast, and cloned and sequenced the corresponding gene encoding a 387‐amino acid protein from Saccharomyces cerevisiae. Gene disruption experiments indicated that the deoxyhypusine synthase gene is essential for cell growth in yeast. This gene was shown to be an intron‐free, single‐copy gene, and its product can catalyze the synthesis of deoxyhypusine equally in two precursor forms of eIF‐5A, derived from two distinct genes of yeast.

Elongation factor 1α is a component of the subcortical actin bundles of characean algae.

Antibodies to elongation factor 1α (EF1α), a known 50 kDa actin‐bundling protein in Dictyostelium, identified a protein in a whole cell extract of the characean alga Nitella pseudoflabellata that had an apparent molecular weight of 51 kDa. Indirect immunofluorescence microscopy revealed labelling by the EF1α antibodies of the subcortical actin bundles, even after the motile organelles of the endoplasm were removed by perfusion with ATP‐containing solutions.

A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3☆

We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3s binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.

Characterization of blasticidin S — resistant mutants of Saccharomyces cerevisiae

SummaryBlasticidin S-resistant mutants of S. cerevisiae were isolated and characterized. Resistant mutations were found to fall into two complementation groups. A single recessive nuclear gene was responsible for each group, donated as bls1 and bls2, respectively. A gene bls1 was linked to an ilv3 gene located on the right arm of chromosome X. The resistant phenotypes from both genes were not associated with ribosomes known to be target sites of Blasticidin S, when analyzed by poly(U)-directed polyphenylalanine synthesis. The resistant mechanisms of the mutations are discussed in this paper.

Isolation of gram quantities of isoleucyl‐tRNA synthetase from an overproducing strain of Escherichia coli and its use for purification of cognate tRNA

The ileS gene coding for isoleucyl‐tRNA synthetase was cloned on a runaway‐replication plasmid. From the cells harboring the plasmid, gram quantities of the synthetase were isolated using two column procedures. The synthetase was used for the purification of cognate tRNA. Isoleucine tRNAGAU of greater than 90% purity was easily isolated by taking advantage of a specific complex formation of the synthetase with cognate tRNA.

Novel Features of the Functional Site and Expression of the Yeast Deoxyhypusine Synthase

A unique amino acid, hypusine, is formed posttranslationally in the precursor of eukaryotic translation initiation factor 5A (eIF-5A). Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine. We reported earlier that the DYS1 gene encoding deoxyhypusine synthase is essential for cell viability and proliferation in yeast. Here, we show by deletion studies that both N- and C-terminal regions, which are not so well conserved, are necessary for the activity of the yeast enzyme. Of the seven cysteine residues present in the yeast enzyme, only one cysteine (position 252; C252) appeared to be essential for its activity. Moderate overexpression of DYS1 showed very little effects on cell growth and no obvious effects on the intracellular level of eIF-5A. However, repression of the expression of DYS1 resulted in near-complete depletion of eIF-5A 24 h after the initiation of repression and was followed by cell growth arrest after another 24 h. This novel finding suggests that the major role of deoxyhypusine synthase in cell proliferation is mediated not only through its modification of the eIF-5A precursor, but also through its regulation of intracellular eIF-5A levels.

Functional Role and Biochemical Properties of Yeast Peptide Elongation Factor 3 (EF-3)

The eukaryotic peptide elongation cycle is well known to be driven by the two complementary factors EF-lα and EF-2, functionally analogous to the bacterial EF-Tu and EF-G, respectively, and the two GTP hydrolysis steps catalyzed by those factors have been considered to be essential for the cycle to run (Kaziro, 1978; Moldave, 19 85). On yeast ribosomes, however, the elongation process additionally requires the third soluble factor, which was found by Skogerson and collaborators (1976, 1977) using poly- (U)-dependent protein synthesis systems and designated as EF-3. Inhibition experiments with monoclonal (Hutchison et al., 1984) and polyclonal (Dasmahapatra and Chakraburtty, 1981; Miyazaki and Kagi-yama, to be published) antibody raised against EF-3 demonstrated that the factor was essential for the elongation phase in the translation of natural mRNA as well as poly(U). A temperature- sensitive yeast mutant producing a thermolabile EF-3 was blocked in the elongation cycle at a non-permissive temperature, indicating the factor indispensable for the in vivo translation (Herrera et al., 1984; Kamath and Chakraburtty, 1986b; Qin et al., 1987).