Meiotic spindle pole bodies acquire the ability to assemble the spore plasma membrane by sequential recruitment of sporulation-specific components in fission yeast

The spindle pole body (SPB) of Schizosaccharomyces pombe is required for assembly of the forespore membrane (FSM) during meiosis. Before de novo biogenesis of the FSM, the meiotic SPB forms outer plaques, an event referred to as SPB modification. A constitutive SPB component, Spo15, plays an indispensable role in SPB modification and sporulation. Here, we analyzed two sporulation-specific genes, spo13(+) and spo2(+), which are not required for progression of meiotic nuclear divisions, but are essential for sporulation. Spo13 is a 16-kDa coiled-coil protein, and Spo2 is a 15-kDa nonconserved protein. Both Spo13 and Spo2 specifically associated with the meiotic SPB. The respective deletion mutants are viable, but defective in SPB modification and in the onset of FSM formation. Spo13 and Spo2 localized on the cytoplasmic side of the SPB in close contact with the nascent FSM. Localization of Spo13 to the SPB was dependent on Spo15 and Spo2; that of Spo2 depended only on Spo15, suggesting that their recruitment to the SPB is strictly controlled. Spo2 physically associated with both Spo15 and Spo13, but Spo13 and Spo15 did not interact directly. Taken together, these observations indicate that Spo2 is recruited to the SPB during meiosis and then assists in the localization of Spo13 to the outer surface of the SPB.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Sugar-binding properties of VIP36, an intracellular animal lectin operating as a cargo receptor

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of approximately 6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.     

Interactions among yeast protein-disulfide isomerase proteins and endoplasmic reticulum chaperone proteins influence their activities

We previously reported that the reductive activities of yeast protein-disulfide isomerase (PDI) family proteins did not completely explain their contribution to the viability of Saccharomyces cerevisiae (Kimura, T., Hosoda, Y., Kitamura, Y., Nakamura, H., Horibe, T., and Kikuchi, M. (2004) Biochem. Biophys. Res. Commun. 320, 359-365). In this study, we examined oxidative refolding activities and found that Mpd1p, Mpd2, and Eug1p exhibit activities of 13.8, 16.0, and 2.16%, respectively, compared with Pdi1p and that activity for Eps1p is undetectable. In analyses of interactions between yeast PDI proteins and endoplasmic reticulum molecular chaperones, we found that Mpd1p alone does not have chaperone activity but that it interacts with and inhibits the chaperone activity of Cne1p, a homologue of mammalian calnexin, and that Cne1p increases the reductive activity of Mpd1p. These results suggest that the interface between Mpd1p and Cne1p is near the peptide-binding site of Cne1p. In addition, Eps1p interacts with Pdi1p, Eug1p, Mpd1p, and Kar2p with dissociation constants (KD) in the range of 10(-7) to 10(-6). Interestingly, co-chaperone activities were completely suppressed in Eps1p-Pdi1p and Eps1p-Mpd1p complexes, although only Eps1p and Pdi1p have chaperone activity. The in vivo consequences of these results are discussed.     

Treatment with leucine stimulates the production of hepatocyte growth factor in vivo

Hepatocyte growth factor (HGF) has pleiotropic effects. Up-regulation of HGF activity in vivo may be beneficial. Branched-chain amino acids (BCAAs) are known to modulate various cellular functions. When starved rats received intraperitoneal injections of valine, leucine or isoleucine, only leucine treatment increased both hepatic and circulating levels of HGF in a dose-dependent manner, up to 1.5 and 2.3 times higher, respectively, than in controls. When young growing rats with free access to food were injected with leucine once a day for a week, HGF levels and liver weights were significantly higher than those of control rats. Furthermore, 1 week of leucine treatment of adult rats resulted in elevated serum albumin levels with an increase in HGF levels. Taken together with our previous report showing that leucine stimulates HGF production by hepatic stellate cells in culture, leucine, among BCAAs, may induce an increase in HGF production by the liver in vivo.     

Cytotoxic screening of medicinal and edible plants in Okinawa,Japan, and identification of the main toxic constituent of Rhodea japonica (Omoto)

The cytotoxic activity of ethanol extracts from 53 parts of 36 species of medicinal and edible plants cultivated in Okinawa was measured by using K562 human leukemia cells by a flow cytometric method. Two extracts from Rhodea japonica and Hypericum chinense were cytotoxic at a concentration of 10 microg/ml. The main cytotoxic constituent of Rhodea japonica was isolated and identified to be rhodexin A, which has been isolated as a cardetonic agent of the plant. The IC(50) value for rhodexin A against the growth of K562 cells was 19 nM, this activity being much stronger than that of ouabain (IC(50), 60 nM).