Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway

To cope with life-threatening high osmolarity, yeast activates the high-osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase cascade. Activated Hog1 regulates the cell cycle, protein translation, and gene expression. Upstream of the HOG pathway are functionally redundant SLN1 and SHO1 signaling branches. However, neither the osmosensor nor the signal generator of the SHO1 branch has been clearly defined. Here, we show that the mucin-like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors for the SHO1 branch. Hyperactive forms of Hkr1 and Msb2 can activate the HOG pathway only in the presence of Sho1, whereas a hyperactive Sho1 mutant activates the HOG pathway in the absence of both Hkr1 and Msb2, indicating that Hkr1 and Msb2 are the most upstream elements known so far in the SHO1 branch. Hkr1 and Msb2 individually form a complex with Sho1, and, upon high external osmolarity stress, appear to induce Sho1 to generate an intracellular signal. Furthermore, Msb2, but not Hkr1, can also generate an intracellular signal in a Sho1-independent manner.     

Loss of calcineurin homologous protein-1 in chicken B lymphoma DT40 cells destabilizes Na+/H+ exchanger isoform-1 protein

NHE1/SLC9A1 is a ubiquitous isoform of vertebrate Na⁺/H⁺ exchangers (NHEs) functioning in maintaining intracellular concentrations of Na⁺ and H⁺ ions. Calcineurin homologous protein-1 (CHP1) binds to the hydrophilic region of NHE1 and regulates NHE1 activity but reportedly does not play a role in translocating NHE1 from the endoplasmic reticulum to the plasma membrane. However, an antiport function of NHE1 requiring CHP1 remains to be clarified. Here we established CHP1-deficient chicken B lymphoma DT40 cells by gene targeting to address CHP1 function. CHP1-deficient cells showed extensive decreases in Na⁺/H⁺ activities in intact cells. Although NHE1 mRNA levels were not affected, NHE1 protein levels were significantly reduced not only in the plasma membrane but in whole cells. The expression of a CHP1 transgene in CHP1-deficient cells rescued NHE1 protein expression. Expression of mutant forms of CHP1 defective in Ca²⁺ binding or myristoylation also partially decreased NHE1 protein levels. Knockdown of CHP1 also caused a moderate decrease in NHE1 protein in HeLa cells. These data indicate that CHP1 primarily plays an essential role in stabilization of NHE1 for reaching of NHE1 to the plasma membrane and its exchange activity. membrane protein; transporter; antiporter; quality control; degradation     

Flavonoids induce germination of basidiospores of the ectomycorrhizal fungus <Emphasis Type="Italic">Suillus bovinus</Emphasis>

Under laboratory conditions, spores of ectomycorrhizal fungi usually germinate very poorly or not at all. In a previous study, we showed that spores of the ectomycorrhizal fungus Suillus bovinus germinated through the combination of activated charcoal treatment of media and co-culture with seedlings of Pinus densiflora, which suggested that some substances contained in root exudates induced the germination. Among the compounds reported from root exudates, flavonoids have been elucidated to play various and substantial roles in plant–microbe interactions; we therefore investigated the effects of flavonoids on basidiospore germination of S. bovinus by the diffusion gradient assay on water agar plates pretreated with charcoal powder. Seven out of the 11 flavonoids tested, hesperidin, morin, rutin, quercitrin, naringenin, genistein, and chrysin, had greater effects than controls, whereas flavone, biochanin A, luteolin, and quercetin showed no positive effects. The effective concentration presumably corresponded to several micromolar levels, which was equivalent to those effective for pollen development, nod gene induction, and spore germination of F. solani f. sp. pisi and AM fungi. The results suggest that flavonoids play a role as signaling molecules in symbiotic relationships between woody plants and ectomycorrhizal fungi.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

Efficient Production of 2,5-Diketo-d-Gluconate via Heterologous Expression of 2-Ketogluconate Dehydrogenase in Gluconobacter japonicus

2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate for d-tartrate and also vitamin C production. Although Gluconobacter oxydans NBRC3293 produces 2,5DKG from d-glucose via d-gluconate and 2-keto-d-gluconate (2KG), with accumulation of the product in the culture medium, the efficiency of 2,5DKG production is unsatisfactory because there is a large amount of residual d-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochrome c complex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied the kgdSLC genes encoding 2KGDH in G. oxydans NBRC3293 to improve 2,5DKG production by Gluconobacter spp. The kgdS, kgdL, and kgdC genes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. The kgdSLC genes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene of G. oxydans for expression in Gluconobacter spp. According to our results, 2KGDH that was purified from the recombinant Gluconobacter cells showed characteristics nearly the same as those reported previously. We also expressed the kgdSLC genes in a mutant strain of Gluconobacter japonicus NBRC3271 (formerly Gluconobacter dioxyacetonicus IFO3271) engineered to produce 2KG efficiently from a mixture of d-glucose and d-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose and d-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of a Gluconobacter strain that produces 2,5DKG efficiently and homogeneously.     

Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1

Axillary shoot formation is a key determinant of plant architecture. Formation of the axillary shoot is regulated by initiation of the axillary meristem or outgrowth of the axillary bud. Here, we show that rice (Oryza sativa) TILLERS ABSENT1 (TAB1; also known as Os WUS), an ortholog of Arabidopsis thaliana WUS, is required to initiate axillary meristem development. We found that formation of the axillary meristem in rice proceeds via a transient state, which we term the premeristem, characterized by the expression of OSH1, a marker of indeterminate cells in the shoot apical meristem. In the tab1-1 (wus-1) mutant, however, formation of the axillary meristem is arrested at various stages of the premeristem zone, and OSH1 expression is highly reduced. TAB1/WUS is expressed in the premeristem zone, where it shows a partially overlapping pattern with OSH1. It is likely, therefore, that TAB1 plays an important role in maintaining the premeristem zone and in promoting the formation of the axillary meristem by promoting OSH1 expression. Temporal expression patterns of WUSCHEL-RELATED HOMEOBOX4 (WOX4) indicate that WOX4 is likely to regulate meristem maintenance instead of TAB1 after establishment of the axillary meristem. Lastly, we show that the prophyll, the first leaf in the secondary axis, is formed from the premeristem zone and not from the axillary meristem.     

Selective, high conversion of D-glucose to 5-keto-D-gluoconate by Gluconobacter suboxydans

Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5-4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity.     

Small molecular CD4 mimics as HIV entry inhibitors

Derivatives of CD4 mimics were designed and synthesized to interact with the conserved residues of the Phe43 cavity in gp120 to investigate their anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. Significant potency gains were made by installation of bulky hydrophobic groups into the piperidine moiety, resulting in discovery of a potent compound with a higher selective index and CD4 mimicry. The current study identified a novel lead compound 11 with significant anti-HIV activity and lower cytotoxicity than those of known CD4 mimics.     

A mitochondrial ubiquitin ligase MITOL controls cell toxicity of polyglutamine-expanded protein

Expansion of a polyglutamine tract in ataxin-3 (polyQ) causes Machado–Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation. Several lines of evidence demonstrate that polyQ also accumulates in mitochondria and causes mitochondrial dysfunction. To uncover the mechanism of mitochondrial quality-control via the ubiquitin–proteasome pathway, we investigated whether MITOL, a novel mitochondrial ubiquitin ligase localized in the mitochondrial outer membrane, is involved in the degradation of pathogenic ataxin-3 in mitochondria. In this study, we used N-terminal-truncated pathogenic ataxin-3 with a 71-glutamine repeat (ΔNAT-3Q71) and found that MITOL promoted ΔNAT-3Q71 degradation via the ubiquitin–proteasome pathway and attenuated mitochondrial accumulation of ΔNAT-3Q71. Conversely, MITOL knockdown induced an accumulation of detergent-insoluble ΔNAT-3Q71 with large aggregate formation, resulting in cytochrome c release and subsequent cell death. Thus, MITOL plays a protective role against polyQ toxicity, and thereby may be a potential target for therapy in polyQ diseases. Our findings indicate a protein quality-control mechanism at the mitochondrial outer membrane via a MITOL-mediated ubiquitin–proteasome pathway.