Hideyuki Hayashi

OCAM: A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons

Zone-to-zone projection of olfactory and vomeronasal sensory axons underlies the topographic and functional mapping of chemoreceptor expression zones of the sensory epithelia onto zonally arranged glomeruli in the main and accessory olfactory bulbs. Here we identified OCAM (R4B12 antigen), an axonal surface glycoprotein expressed by subsets of both olfactory and vomeronasal axons in a zone-specific manner. OCAM is a novel homophilic adhesion molecule belonging to the immunoglobulin superfamily with striking structural homology to neural cell adhesion molecule. In both the main and accessory olfactory systems, OCAM mRNA is expressed by sensory neurons in restricted chemoreceptor expression zones, and OCAM protein-expressing axons project to the glomeruli in the corresponding zones of the main and accessory bulbs. OCAM protein is expressed on subsets of growing sensory axons in explant cultures even in the absence of the target bulb. These results demonstrate a precisely coordinated zonal expression of chemoreceptors and OCAM and suggest that OCAM may play important roles in selective fasciculation and zone-to-zone projection of the primary olfactory axons.

Yeast Nfs1p Is Involved in Thio-modification of Both Mitochondrial and Cytoplasmic tRNAs

The IscS protein is a pyridoxal phosphate-containing cysteine desulfurase involved in iron-sulfur cluster biogenesis. In prokaryotes, IscS is also involved in various metabolic functions, including thio-modification of tRNA. By contrast, the eukaryotic ortholog of IscS (Nfs1) has thus far been shown to be functional only in mitochondrial iron-sulfur cluster biogenesis. We demonstrate here that yeast Nfs1p is also required for the post-transcriptional thio-modification of both mitochondrial (mt) and cytoplasmic (cy) tRNAs in vivo. Depletion of Nfs1p resulted in an immediate impairment of the 2-thio-modification of 5-carboxymethylaminomethyl-2-thiouridine at the wobble positions of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{mt-tRNA}_{\mathrm{UUU}}^{\mathrm{Lys}}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{mt-tRNA}_{\mathrm{UUG}}^{\mathrm{Gln}}\) \end{document}. In addition, we observed a severe reduction in the 2-thio-modification of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{cy-tRNA}_{\mathrm{UUU}}^{\mathrm{Lys}2}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{cy-tRNA}_{\mathrm{UUC}}^{\mathrm{Glu}3}\) \end{document}, although the effect was somewhat delayed compared with that seen in mt-tRNAs. Mass spectrometry analysis revealed an increase in 5-methoxycarbonylmethyluridine concomitant with a decrease in mcm5s2U in cy-tRNAs that were prepared from Nfs1p-depleted cells. These results suggest that Nfs1p is involved in the 2-thio-modification of both 5-carboxymethylaminomethyl-2-thiouridine in mt-tRNAs and mcm5s2U in cy-tRNAs.

Nuclear Localization of Yeast Nfs1p Is Required for Cell Survival

Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.

Thio-modification of Yeast Cytosolic tRNA Requires a Ubiquitin-related System That Resembles Bacterial Sulfur Transfer Systems

The wobble uridine in yeast cytosolic tRNALys2UUU and tRNAGlu3UUC undergoes a thio-modification at the second position (s2 modification) and a methoxycarbonylmethyl modification at the fifth position (mcm5 modification). We previously demonstrated that the cytosolic and mitochondrial iron-sulfur (Fe/S) cluster assembly machineries termed CIA and ISC, including a cysteine desulfurase called Nfs1, were essential for the s2 modification. However, the cytosolic component that directly participates in this process remains unclear. We found that ubiquitin-like protein Urm1 and ubiquitin-activating enzyme-like protein Uba4, as well as Tuc1 and Tuc2, were strictly required for the s2 modification. The carboxyl-terminal glycine residue of Urm1 was critical for the s2 modification, indicating direct involvement of the unique ubiquitin-related system in this process. We also demonstrated that the s2 and mcm5 modifications in cytosolic tRNAs influence each others efficiency. Taken together, our data indicate that the s2 modification of cytosolic tRNAs is a more complex process that requires additional unidentified components.

Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration.

Aureusidin synthase, which plays a key role in the yellow coloration of snapdragon flowers, is a homolog of plant polyphenol oxidase (PPO). The enzyme specifically acted on chalcones with a 4‐monohydroxy or 3,4‐dihydroxy B‐ring to produce aurones, for whose production the oxidative cyclization of chalcones must be preceded by 3‐oxygenation. However, it exhibited virtually no PPO activity toward non‐chalcone phenolics. The enzyme was competitively inhibited by phenylthiourea, a specific PPO inhibitor. These results led us to propose a mechanism of aurone synthesis by aureusidin synthase on the basis of known PPO‐catalyzed reactions and conclude that the enzyme is a chalcone‐specific PPO specialized for aurone biosynthesis.

Activation of protein phosphatase 2A by cAMP‐dependent protein kinase‐catalyzed phosphorylation of the 74‐kDa B″ (δ) regulatory subunit in vitro and identification of the phosphorylation sites

Human erythrocyte protein phosphatase 2A, which comprises a 34‐kDa catalytic C subunit, a 63‐kDa regulatory A subunit and a 74‐kDa regulatory B″ (δ) subunit, was phosphorylated at serine residues of B″ in vitro by cAMP‐dependent protein kinase (A‐kinase). In the presence and absence of 0.5 μM okadaic acid (OA), A‐kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B″, respectively. The K m value of A‐kinase for CAB″ was 0.17±0.01 μM in the presence of OA. The major in vitro phosphorylation sites of B″ were identified as Ser‐60, ‐75 and ‐573 in the presence of OA, and Ser‐75 and ‐573 in the absence of OA. Phosphorylation of B″ did not dissociate B″ from CA, and stimulated the molecular activity of CAB″ toward phosphorylated H1 and H2B histones, 3.8‐ and 1.4‐fold, respectively, but not toward phosphorylase a.

Thio Modification of Yeast Cytosolic tRNA Is an Iron-Sulfur Protein-Dependent Pathway†

ABSTRACT Defects in the yeast cysteine desulfurase Nfs1 cause a severe impairment in the 2-thio modification of uridine of mitochondrial tRNAs (mt-tRNAs) and cytosolic tRNAs (cy-tRNAs). Nfs1 can also provide the sulfur atoms of the iron-sulfur (Fe/S) clusters generated by the mitochondrial and cytosolic Fe/S cluster assembly machineries, termed ISC and CIA, respectively. Therefore, a key question remains as to whether the biosynthesis of Fe/S clusters is a prerequisite for the 2-thio modification of the tRNAs in both of the subcellular compartments of yeast cells. To elucidate this question, we asked whether mitochondrial ISC and/or cytosolic CIA components besides Nfs1 were involved in the 2-thio modification of these tRNAs. We demonstrate here that the three CIA components, Cfd1, Nbp35, and Cia1, are required for the 2-thio modification of cy-tRNAs but not of mt-tRNAs. Interestingly, the mitochondrial scaffold proteins Isu1 and Isu2 are required for the 2-thio modification of the cy-tRNAs but not of the mt-tRNAs, while mitochondrial Nfs1 is required for both 2-thio modifications. These results clearly indicate that the 2-thio modification of cy-tRNAs is Fe/S protein dependent and thus requires both CIA and ISC machineries but that of mt-tRNAs is Fe/S cluster independent and does not require key mitochondrial ISC components except for Nfs1.

A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction.

Serine palmitoyltransferase (SPT, EC 2.3.1.50) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395T have significant SPT activity and purified SPT to homogeneity. This enzyme is a water-soluble homodimeric protein unlike eukaryotic enzymes, known as heterodimers composed of tightly membrane-bound subunits, named LCB1 and LCB2. The purified SPT shows an absorption spectrum characteristic of a pyridoxal 5′-phosphate-dependent enzyme. The substrate specificity of theSphingomonas SPT is less strict than the SPT complex from Chinese hamster ovary cells. We isolated the SPT gene encoding 420 amino acid residues (M r 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10−20% of the total protein of the cell extract. Sphingomonas SPT shows about 30% homology with the enzymes of the α-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The recombinant SPT was catalytically and spectrophotometrically indistinguishable from the native enzyme. This is the first successful overproduction of an active enzyme in the sphingolipid biosynthetic pathway. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.

Molecular cloning of a 74-kDa regulatory subunit (B″ or δ) of human protein phosphatase 2A

Based on amino acid sequence data of a 74‐kDa regulatory subunit (B″ or δ) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an M r 66 138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74‐kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, an SH3 accessible proline‐rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

[Arg292----Val] or [Arg292----Leu] mutation enhances the reactivity of Escherichia coli aspartate aminotransferase with aromatic amino acids.

Arg292 of E. coli aspartate aminotransferase was substituted with valine or leucine by site-directed mutagenesis. In comparison with the wild-type enzyme, either of the mutant enzymes showed a decrease by over 5 orders of magnitude of kcat/km values for aspartate and glutamate. This supports the contention that Arg292 is important for determining the specificity of this enzyme for dicarboxylic substrates. In contrast, mutant enzymes displayed a 5- to 10-fold increase in kcat/Km values for aromatic amino acids as substrates. Thus, introduction of an uncharged, hydrophobic side chain into position 292 leads to a striking alteration in substrate specificity of this enzyme, thereby improving catalytic efficiency toward aromatic amino acids.