ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase

In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by N?rby et al. (N?rby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P----E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+.     

Regeneration in subalpine coniferous forests

Death of canopy trees when gaps are formed was studied in a subalpine coniferous forest, central Japan, which was composed ofAbies, Tsuga, Picea, Betula, andSorbus. Typhoons were considered to be the most important cause of the death of canopy trees. The degree of disturbance in each of 16 plots (20 m×20 m) was represented by the percentage of the total basal area of dead trees to that of living and dead canopy trees (disturbance magnitude; MAG). The mortality of canopy trees increased as their dbh increase in the plots of lower MAG than 90%. The mortality varied among genera, andTsuga was characterized as having lower mortality than that of the other conifers. 418 dead trees were observed. The standing dead trees made up 10.7% of the trees, the stem broken trees 46.7%, and the uprooted trees 42.2%. The stem breaking was most frequent inAbies, and the uprooting was most frequent inTsuga, Picea, andBetula. Undeveloped forests, which have the L-shaped dbh distribution, were destroyed only in high degree (70%<MAG), while developed forests were destroyed in various degrees (30%<MAG<100%). The percentage of uprooted trees in basal area decreased with the development of the forest, from 60% to 10%.     

Climate and vegetation in China V. Effect of climatic factors on the upper limit of distribution of evergreen broadleaf forest

In order to clarify correlations between the upper limit of distribution of evergreen broadleaf forest and climatic factors, 62 stands distributed at the upper limit of the forest were collected from various parts of China, and their thermal and precipitation factors were estimated. Among six thermal climatic indices, i.e., warmth index (WI), coldness index (CI) and annual mean (AMT), January mean (JMT), mean minimum (MMT) and minimum (MT) temperatures, the CI at the stands showed the smallest range of variance, and it was therefore considered to be the most significant for interpreting the upward distribution of the forest. However, the distribution of the forest in mountain areas in southwestern China could not be explained by lower temperatures in winter such as CI but by a cumulative temperature such as WI. The continentality and precipitation factors were also important for delimiting the distribution of the forest. In addition, the relation between the distribution of the forest and the MMT was noted, and it was concluded that the MMT was an effective thermal index for explaining the upper limit of distribution of evergreen broadleaf forest in China.     

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.     

Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [gamma-32P]ATP revealed that 7% of the DNA fragments were covalently linked with mono- to octaribonucleotide primers at their 5' ends. Octaribonucleotides, the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [alpha-32P]GTP, indicating that they were the intact primer RNA with tri- (or di-) phosphate termini, and the shorter ribooligomers were degradation intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were rich in A.     

Two different phosphorylation-dephosphorylation cycles of Na,K-ATPase proteoliposomes accompanying Na+ transport in the absence of K+

The phosphorylated intermediate (EP) of the Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme is composed of an ADP-sensitive K+-insensitive form (E1P), an ADP- and K+-sensitive form (E*P), and a K+-sensitive ADP-insensitive form (E2P). The composition of the intermediate varied with the cholesterol content of the lipid bilayer. The PL containing less than 30 mol % cholesterol (LCPL) formed E2P-rich EP in the presence of 10 mM Na+ on both sides at 15 degrees C, while the PL containing more than 35 mol % cholesterol (HCPL) formed E*P-rich EP under the same condition. In the presence of ionophore (monensin, nigericin, A23187), the HCPL formed E2P-rich EP as reported in the preceding paper. The turnover rate of Na-ATPase activity (the ratio of Na-ATPase to the EP level) in the LCPL was lower than that in the HCPL, and the addition of 20 microM monensin or A23187 to the HCPL reduced the Na-ATPase activity. The coupling ratio of Na+ influx (cellular efflux):Na+ efflux (cellular influx):ATP hydrolysis was 2.8:1.8:1 in the LCPL, although 1.6:0.6:1 in the HCPL. The coupling ratio of Na+ influx:ATP hydrolysis in the HCPL increased to 2.8:1 in the presence of A23187. Moreover, the increase of ATP concentration enhanced not only the Na-ATPase activity in the LCPL and HCPL with monensin but also the Na+ influx in the LCPL. This ATP enhancement was not found, however, in the HCPL without ionophores. The ADP enhancement of the Na+ influx was not observed in either the HCPL or the LCPL. We conclude from these observations that there are at least two different phosphorylation-dephosphorylation cycles (an E2P cycle and an E*P cycle) in the PL in the absence of K+. The E2P cycle transports three Na+ from the extravesicular (cytoplasmic) to the intravesicular (extracellular) side and two Na+ in the opposite direction per cycle and is similar to the ATP-dependent Na+-Na+ exchange system already reported (Blostein, R. (1983) J. Biol. Chem. 258, 7948-7953; Cornelius, F., and Skou, J. C. (1985) Biochim. Biophys. Acta 818, 211-221). However, the E*P cycle transports one Na+ from the extravesicular to the intravesicular side/cycle and has not yet been previously reported.     

Action of multiple endoplasmic reticulum chaperon-like proteins is required for proper folding and polarized localization of Kre6 protein essential in yeast cell wall β-1,6-glucan synthesis

Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall β-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for β-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in β-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in β-1,6-glucan synthesis.     

Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene

In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal.