Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR5-like protein in Japanese pear (Pyrus serotina Rehd.)

The stylar proteins of Japanese pear (Pyrus serotina Rehd.) were analyzed by two-dimensional gel electrophoresis, and a 32-kDa protein with an isoelectric point of 4.8 was found to be a major component in the style. The 32-kDa protein was a soluble glycoprotein which reacted with concanavalin A. The 32-kDa protein specifically accumulated in the style in a developmentally regulated manner, but was not detected in the other floral organs and leaves. An oligonucleotide representing the N-terminal amino acid sequence of the 32-kDa protein was used to amplify a cDNA fragment by polymerase chain reaction (PCR). The generated PCR product was used to screen a style cDNA library. The selected cDNA clone encoded 244 amino acid residues containing the N-terminal sequence of the 32-kDa protein. The N-terminus of the protein was preceded by putative signal peptide of 22 amino acid residues. The 32-kDa protein showed significant homology with the thaumatin/PR5-like proteins, and was named PsTL1 (Pyrus serotina thaumatin-like protein 1). The possible biological role of PsTL1 in the styles is discussed. Received: 27 November 1997 / Accepted: 19 January 1998     

The effect on photosynthetic electron transport of temperature-dependent changes in the fluidity of the thylakoid membrane in a thermophilic blue-green alga

Various electron transport reactions in cell or isolated thylakoid membranes of the thermophilic blue-green alga, Synechococcus sp. were measured at different temperatures between 72 and 3 degrees C. They are classified into two groups with respect to their temperature dependency. The first group involves cytochrome 553 photooxidation, methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol as electron donor and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea-resistant ferricyanide photoreduction determined in the presence or absence of silicomolybdate. The Arrhenius plot of these reactions showed a single straight line with the activation energy of about 10 kcal/mol throughout wide temperature ranges studied. Methyl viologen photoreduction with water as electron donor, reduction of flash-oxidized cytochrome 553, ferricyanide photoreduction and photosynthetic O2 evolution form the second group. Their arrhenius plots are characterized by discontinuities or breaks at about 30 and 10 degrees C, which respectively correspond to the upper and lower boundaries of the lateral phase separation of the membrane lipids. The first group reactions represent short spans of electron transport which are mediated either by Photosystem I or Photosystem II alone and not related to plastoquinone, whereas all the reactions of the second group involve plastoquinone. It is concluded therefore that the membrane fluidity affect electron transport specifically at the region of plastoquinone. It is proposed that the reaction center chlorophyll-protein complexes of both Photosystems I and II are closely associated with related electron carrier proteins to form functional supramolecular assemblies so that electron transfer within such a cluster of proteins proceeds independently of the phase changes in the membrane lipids. On the other hand, the role of plastoquinone as a mobile electron carrier mediating electron transfer from the protein assembly of Photosystem II to that of Photosystem I through the fluid hydrophobic matrix of the membranes is highly sensitive to the physical state of the membrane lipids.     

Crystal structure of VioE, a key player in the construction of the molecular skeleton of violacein

Violacein and the indolocarbazoles are naturally occurring bisindole products with various biological activities, including antitumor activity. Although these compounds have markedly different molecular skeletons, their biosynthetic pathways share the same intermediate "compound X," which is produced from L-tryptophan via indole-3-pyruvic acid imine. Compound X is a short-lived intermediate that is spontaneously converted to chromopyrrolic acid for indolocarbazole biosynthesis, whereas VioE transforms compound X into protodeoxyviolaceinic acid, which is further modified by other enzymes to produce violacein. Thus, VioE plays a key role in the construction of the molecular skeleton of violacein. Here, we present the crystal structure of VioE, which consists of two subunits, each of which forms a structure resembling a baseball glove. Each subunit has a positively charged pocket at the center of the concave surface of the structure. Mutagenesis analysis of the surface pocket and other surface residues showed that the surface pocket serves as an active site. We have also solved the crystal structure of a complex of VioE and phenylpyruvic acid as an analogue of a VioE-substrate complex. A docking simulation with VioE and the IPA imine dimer, which is proposed to be compound X, agreed with the results from the mutational analysis and the VioE-phenylpyruvic acid complex structure. Based on these results, we propose that VioE traps the highly reactive substrate within the surface pocket to suppress CPA formation and promote protodeoxyviolaceinic acid formation caused by proximity and orientation effects.     

Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction

OL-protocadherin (OL-pc) is a transmembrane protein belonging to the cadherin superfamily, which has been shown to accumulate at cell-cell contacts via its homophilic interaction, but its molecular roles remain elusive. In this study, we show that OL-pc bound Nck-associated protein 1 (Nap1), a protein that regulates WAVE-mediated actin assembly. In astrocytoma U251 cells not expressing OL-pc, Nap1 was localized only along the lamellipodia. However, exogenous expression of OL-pc in these cells recruited Nap1 as well as WAVE1 to cell-cell contact sites. Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions. OL-pc mutants lacking the Nap1-binding site exhibited no such effect. N-cadherin knockdown mimicked OL-pc expression in enhancing cell movement. These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1-WAVE1 complex to these sites and, in turn, promotes the migration of cells.     

ADCK3, an ancestral kinase, is mutated in a form of recessive ataxia associated with coenzyme Q10 deficiency

Muscle coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency has been identified in more than 20 patients with presumed autosomal-recessive ataxia. However, mutations in genes required for CoQ(10) biosynthetic pathway have been identified only in patients with infantile-onset multisystemic diseases or isolated nephropathy. Our SNP-based genome-wide scan in a large consanguineous family revealed a locus for autosomal-recessive ataxia at chromosome 1q41. The causative mutation is a homozygous splice-site mutation in the aarF-domain-containing kinase 3 gene (ADCK3). Five additional mutations in ADCK3 were found in three patients with sporadic ataxia, including one known to have CoQ(10) deficiency in muscle. All of the patients have childhood-onset cerebellar ataxia with slow progression, and three of six have mildly elevated lactate levels. ADCK3 is a mitochondrial protein homologous to the yeast COQ8 and the bacterial UbiB proteins, which are required for CoQ biosynthesis. Three out of four patients tested showed a low endogenous pool of CoQ(10) in their fibroblasts or lymphoblasts, and two out of three patients showed impaired ubiquinone synthesis, strongly suggesting that ADCK3 is also involved in CoQ(10) biosynthesis. The deleterious nature of the three identified missense changes was confirmed by the introduction of them at the corresponding positions of the yeast COQ8 gene. Finally, a phylogenetic analysis shows that ADCK3 belongs to the family of atypical kinases, which includes phosphoinositide and choline kinases, suggesting that ADCK3 plays an indirect regulatory role in ubiquinone biosynthesis possibly as part of a feedback loop that regulates ATP production.     

A simple and precise method for measuring HDL-cholesterol subfractions by a single precipitation followed by homogenous HDL-cholesterol assay

HDL consists of two major subfractions, HDL2 and HDL3. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl2) and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL3-C was measured by a homogenous HDL-C assay in the supernatant, and HDL2-C was estimated by subtracting the HDL3-C from the direct HDL-C. The HDL3-C and HDL2-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL3-C and HDL2-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory.