Involvement of Golgi-associated retrograde protein complex in the recycling of the putative Dnf aminophospholipid flippases in yeast

It is widely accepted that phosphatidylethanolamine (PE) is enriched in the cytosolic leaflet of the eukaryotic plasma membranes. To identify genes involved in the establishment and regulation of the asymmetric distribution of PE on the plasma membrane, we screened the deletion strain collection of the yeast Saccharomyces cerevisiae for hypersensitive mutants to the lantibiotic peptide Ro09-0198 (Ro) that specifically binds to PE on the cell surface and inhibits cellular growth. Deletion mutants of VPS51, VPS52, VPS53, and VPS54 encoding the components of Golgi-associated retrograde protein (GARP) complex, YPT6 encoding a Rab family small GTPase that functions with GARP complex, RIC1 and RGP1 encoding its guanine nucleotide exchange factor (GEF), and TLG2 encoding t-SNARE exhibited hypersensitivity to Ro. The mutants deleted for VPS51, VPS52, VPS53, and VPS54 were impaired in the uptake of fluorescently labeled PE. In addition, aberrant intracellular localization of the EGFP-tagged Dnf2p, the putative inward-directed phospholipid translocase (flippase) of the plasma membrane, was observed in the mutant defective in the GARP complex, Ypt6p, its GEF proteins, or Tlg2p. Our results suggest that the GARP complex is involved in the recycling of Dnf flippases.     

Malting quality quantitative trait loci on a high-density map of Mikamo golden?×?Harrington cross in barley (Hordeum vulgare L.)

A high-density map consisting of 550 markers was constructed based on the segregation data of 95 doubled-haploid lines (DHLs) derived from the cross between a Japanese barley cultivar, Mikamo Golden and a North American barley cultivar, Harrington (MH-DHLs). Quality traits of malt extract (EX), total nitrogen (TN), soluble nitrogen (SN), Kolbach index (KI), diastatic power (DP), wort beta-glucan (WG) and viscosity (VS) were determined in three site/year crops. Quantitative trait loci (QTL) analyses were performed with these quality data sets, using the linkage map. Major QTL controlling EX, SN and KI were mapped on terminal region of 5H with Harrington as effective allele. Another QTL controlling EX was mapped on 2H with Mikamo Golden as effective allele. QTL controlling TN, DP, WG and VS were detected variably in terms of flanking markers and chromosomes depending on site/year. Cleaved amplified polymorphic sequences (CAPS) markers for EX based on the QTL detected on 2H and 5H were developed. Analysis of EX and genotypes of 33 malting barley cultivars from around the world as well as MH-DHLs revealed that the two CAPS marker on 2H and 5H affect EX by a significant difference, suggesting that the two CAPS markers were valuable for marker-assisted selection in malting barley breeding.     

Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae Δpem1 Δpem2 double mutant without exogenous choline supply

In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3 × HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations.     

Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana

The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de-etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a: b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full-green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light-harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green-gel system increased by 20%. (3) The ratio of the light-harvesting complex II apoproteins (LHCP) to 47-kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10-20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.     

Development of an orexin-2 receptor selective agonist, [Ala(11), D-Leu(15)]orexin-B

Investigation of L-alanine and D-amino acid replacement of orexin-B revealed that three L-leucine residues at the positions of 11, 14, and 15 in orexin-B were important to show selectivity for the orexin-2 receptor (OX(2)) over the orexin-1 receptor (OX(1)). L-Alanine substitution at position 11 and D-leucine substitution at positions 14 and 15 maintained the potency of orexin-B to mobilize [Ca(2+)](i) in CHO cells expressing the OX(2), while their potency for the OX(1) was significantly reduced. In combined substitutions, we identified that [Ala(11), D-Leu(15)]orexin-B showed a 400-fold selectivity for the OX(2) (EC(50)=0.13nM) over OX(1) (EC(50)=52nM). [Ala(11), D-Leu(15)]orexin-B is a beneficial tool for addressing the functional roles of the OX(2).     

Two new species of the interstitial genus Parvocythere (Crustacea, Ostracoda, Cytheroidea) from Japan: an example of morphological variation

Two new species of the interstitial ostracod genus Parvocythere, Parvocythere gottwaldisp. n. and Parvocythere gracilissp. n., are herein described. Although these two new species are clearly distinguishable by certain morphological differences in elements of the male copulatory organ, and the carapace, they share the following simplified characters of the appendages and male copulatory organ: antennular fourth podomere with no suture; reduced claws on the distal end of antenna; and asymmetric male copulatory organ. The morphological differences among known and new Parvocythere species suggest that the species of this genus can be classified into two groups by the presence/absence of the suture on the antennular fourth podomere. The "Group S" is characterised by the presence of the antennular suture, and all species of this group have a two-clawed antenna and symmetric male copulatory organ, characters which are generally seen in cytheroid ostracods. The species belonging to "Group N" are characterised by the absence of the suture, regarded as a pedomorphic character, show the following characters: two clawed or one clawed antenna, and symmetric or asymmetric male copulatory organ. The morphological variation within Group N includes reductive characters regarded as an adaptation to the narrow spaces of the interstitial environment of a sandy beach. These intrageneric morphological variations of the exclusively interstitial genus Parvocythere suggest the possibilities that Group N might be derived from Group S, and that some adaptive characters to an interstitial environment could have developed after the colonisation of these environments.     

Absorption of the indigestible disaccharide, β-1,4-mannobiose, from coconut by the rat portal vein

The intestinal absorption of β-1,4-mannobiose by rats was investigated. Mannobiose was detected in the portal vein plasma by matrix-assisted laser desorption ionization/time of flight mass spectrometry after its administration to rats. The presence of mannobiose in the rat plasma was confirmed by an experiment using β-mannosidase. These results indicate that mannobiose was directly absorbed through the intestines even without being hydrolyzed.     

Incorporation and remodeling of phosphatidylethanolamine containing short acyl residues in yeast

Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn− 2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn− 2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.     

The major route for chlorophyll synthesis includes [3,8-divinyl]-chlorophyllide a reduction in Arabidopsis thaliana

In most reviews on Chl biosynthesis, Chl is described as being synthesized via the route involving the reduction of [3,8-divinyl]-protochlorophyllide a. However, the possibility remains that the conversion of the divinyl form of the Chl intermediate to its monovinyl form takes place at other enzymatic steps. To determine the actual route of Chl biosynthesis, we examined the substrate specificity of the formerly named [3,8-divinyl]-protochlorophyllide a 8-vinyl reductase (DVR) in vitro. In addition, we investigated the accumulation of various Chl intermediates in etiolated seedlings in vivo. Collectively, these studies indicate that [3,8-divinyl]-chlorophyllide a is the major substrate of DVR.