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Yasuyoshi Sakai

Publications and source records attributed to Yasuyoshi Sakai.

At least 19 recordsLinked to original sources
PubMed2007

Functions of PI4P and sterol glucoside are necessary for the synthesis of a nascent membrane structure during pexophagy.

We recently showed that, in the yeast Pichia pastoris, an ergosterol glucoside synthesizing enzyme, Atg26, is recruited to the precursor of the pexophagic structure, micropexophagic membrane apparatus (MIPA), under the regulation of phosphatidylinositol 4'-monophosphate (PI4P)-signaling during pexophagy. Atg26 was found to harbor a novel PI4P-binding motif, the GRAM domain. Both lipids, PI4P and sterol glucoside, synthesized by PpPik1 and PpAtg26, respectively, were necessary for pexophagy, in the step where the MIPA was formed. In this addendum, we review these findings, and speculate on the mechanistic and physiological implications of the functions of these lipids during the autophagic process.

Animals↗
PubMed2006

Role of Vac8 in formation of the vacuolar sequestering membrane during micropexophagy.

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.

Alcohol Oxidoreductases↗
PubMed2006

Novel acetone metabolism in a propane-utilizing bacterium, Gordonia sp. strain TY-5.

In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane --> 2-propanol --> acetone --> methyl acetate --> acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.

Acetone↗
PubMed2006

The significance of peroxisomes in methanol metabolism in methylotrophic yeast.

The capacity to use methanol as sole source of carbon and energy is restricted to relatively few yeast species. This may be related to the low efficiency of methanol metabolism in yeast, relative to that of prokaryotes. This contribution describes the details of methanol metabolism in yeast and focuses on the significance of compartmentalization of this metabolic pathway in peroxisomes.

Alcohol Oxidoreductases↗
PubMed2006

Pexophagy: autophagic degradation of peroxisomes.

The abundance of peroxisomes within a cell can rapidly decrease by selective autophagic degradation (also designated pexophagy). Studies in yeast species have shown that at least two modes of peroxisome degradation are employed, namely macropexophagy and micropexophagy. During macropexophagy, peroxisomes are individually sequestered by membranes, thus forming a pexophagosome. This structure fuses with the vacuolar membrane, resulting in exposure of the incorporated peroxisome to vacuolar hydrolases. During micropexophagy, a cluster of peroxisomes is enclosed by vacuolar membrane protrusions and/or segmented vacuoles as well as a newly formed membrane structure, the micropexophagy-specific membrane apparatus (MIPA), which mediates the enclosement of the vacuolar membrane. Subsequently, the engulfed peroxisome cluster is degraded. This review discusses the current state of knowledge of pexophagy with emphasis on studies on methylotrophic yeast species.

Animals↗
PubMed2006

PI4P-signaling pathway for the synthesis of a nascent membrane structure in selective autophagy.

Phosphoinositides regulate a wide range of cellular activities, including membrane trafficking and biogenesis, via interaction with various effector proteins that contain phosphoinositide binding motifs. We show that in the yeast Pichia pastoris, phosphatidylinositol 4'-monophosphate (PI4P) initiates de novo membrane synthesis that is required for peroxisome degradation by selective autophagy and that this PI4P signaling is modulated by an ergosterol-converting PpAtg26 (autophagy-related) protein harboring a novel PI4P binding GRAM (glucosyltransferase, Rab-like GTPase activators, and myotubularins) domain. A phosphatidylinositol-4-OH kinase, PpPik1, is the primary source of PI4P. PI4P concentrated in a protein-lipid nucleation complex recruits PpAtg26 through an interaction with the GRAM domain. Sterol conversion by PpAtg26 at the nucleation complex is necessary for elongation and maturation of the membrane structure. This study reveals the role of the PI4P-signaling pathway in selective autophagy, a process comprising multistep molecular events that lead to the de novo membrane formation.

1-Phosphatidylinositol 4-Kinase↗
PubMed2006

Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica.

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, P(MOD1) and P(MOD2), were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5) as a reporter. Heterologous gene expression driven by the P(MOD1) and P(MOD2) promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. P(MOD1) was induced not only by methanol but also by glycerol. P(MOD2) was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that P(MOD2) is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by P(MOD1) and P(MOD2). P(MOD1) expression was optimal at low methanol concentrations, whereas P(MOD2) was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both P(MOD2) and P(MOD1) should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using P(MOD1) and P(MOD2) in the same host cell and in the same flask.

Acid Phosphatase↗
PubMed2006

Purification and characterization of benzoate-CoA ligase from Magnetospirillum sp. strain TS-6 capable of aerobic and anaerobic degradation of aromatic compounds.

Benzoate-CoA ligase (EC 6.2.1.25), the initial enzyme of anaerobic benzoate degradation, was purified and characterized from Magnetospirillum sp. strain TS-6 grown under both anaerobic and aerobic conditions. The enzyme purified from anaerobically grown cells was a homodimer with a relative molecular mass of 120 kDa. The specific activity for benzoyl-CoA synthesis was 13.4 micromol min(-1) mg(-1) protein. The enzyme purified from aerobically grown cells was concluded to be the same gene product as the anaerobic enzyme. The benzoate-CoA ligase gene consisting of 1587 nucleotides was cloned and sequenced, and its induction under aerobic and anaerobic conditions during growth on benzoate was confirmed by quantitative reverse transcription PCR. These results indicate that a single benzoate-CoA ligase is expressed and benzoate is converted into benzoyl-CoA under both aerobic and anaerobic conditions in Magnetospirillum sp.

Benzoates↗
PubMed2006

The ribulose monophosphate pathway substitutes for the missing pentose phosphate pathway in the archaeon Thermococcus kodakaraensis.

The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Interestingly, HPS and PHI homologs are also found in a variety of archaeal strains, and recent biochemical and genome analyses have raised the possibility that the reverse reaction of formaldehyde fixation, i.e., ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may function in the biosynthesis of Ru5P in some archaeal strains whose pentose phosphate pathways are imperfect. In this study, we have taken a genetic approach to address this possibility by using the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. This strain possesses a single open reading frame (TK0475) encoding an HPS- and PHI-fused protein. The recombinant HPS-PHI-fused enzyme exhibited the expected HPS and PHI activities in both directions (formaldehyde fixing and Ru5P synthesizing). The TK0475 deletion mutant Delta hps-phi-7A did not exhibit any growth in minimal medium, while growth of the mutant strain could be recovered by the addition of nucleosides to the medium. This auxotrophic phenotype together with the catalytic properties of the HPS-PHI-fused enzyme reveal that HPS and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is the only relevant pathway for Ru5P biosynthesis substituting for the classical pentose phosphate pathway missing in this archaeon.

Aldehyde-Lyases↗
PubMed2006

Gene structure and regulation of alkane monooxygenases in propane-utilizing Mycobacterium sp. TY-6 and Pseudonocardia sp. TY-7.

Mycobacterium sp. TY-6 and Pseudonocardia sp. TY-7 were isolated from soil samples as propane-utilizing bacteria and were found to be able to utilize various gaseous and liquid n-alkanes as carbon and energy sources. One gene cluster, M-prmABCD, and two gene clusters, P-prm1ABCD and P-prm2ABCD, were cloned from the genomes of Mycobacterium sp. TY-6 and Pseudonocardia sp. TY-7, respectively. These gene clusters are homologous to the gene cluster encoding the multicomponent propane monooxygenase (prmABCD) of Gordonia sp. TY-5. The expression of prm gene clusters in Mycobacterium sp. TY-6 and Pseudonocardia sp. TY-7 was shown to be induced by gaseous n-alkanes (C2-C4) except methane, suggesting that the products of these genes are involved in gaseous n-alkane oxidation. Homologous genes for an alkane hydroxylase system (alk system) involved in liquid n-alkane oxidation were also cloned from the genomic DNA of Mycobacterium sp. TY-6. The alk gene cluster was transcribed in response to liquid n-alkanes (C11-C15). These results indicate that Mycobacterium sp. TY-6 has two distinct gene clusters for multicomponent monooxygenases involved in alkane oxidation. Whole-cell reactions revealed that propane is oxidized to 1-propanol through terminal oxidation in Mycobacterium sp. TY-6 and that propane is oxidized to 1-propanol and 2-propanol through both terminal and subterminal oxidations in Pseudonocardia sp. TY-7. This study reveals the diversity of propane metabolism present in microorganisms.

Actinomycetales↗
PubMed2005

Isolation and functional characterization of fatty acid delta5-elongase gene from the liverwort Marchantia polymorpha L.

Bryophyte Marchantia polymorpha L. produces C22 very-long-chain polyunsaturated fatty acid (VLCPUFA). Thus far, no enzyme that mediates elongation of C20 VLCPUFAs has been identified in land plants. Here, we report the isolation and characterization of the gene MpELO2, which encodes an ELO-like fatty acid elongase in M. polymorpha. Heterologous expression in yeast demonstrated that MpELO2 encodes delta5-elongase, which mediates elongation of arachidonic (20:4) and eicosapentaenoic acids (20:5). Phylogenetic and gene structural analysis indicated that the MpELO2 gene is closely related to bryophyte Delta6-elongase genes for C18 fatty acid elongation and diverged from them by local gene duplication.

Acetyltransferases↗
PubMed2005

A front-end desaturase from Chlamydomonas reinhardtii produces pinolenic and coniferonic acids by omega13 desaturation in methylotrophic yeast and tobacco.

Pinolenic acid (PA; 18:3Delta(5,9,12)) and coniferonic acid (CA; 18:4Delta(5,9,12,15)) are Delta(5)-unsaturated bis-methylene-interrupted fatty acids (Delta(5)-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2Delta(9,12)) and alpha-linolenic acid (ALA; 18:3Delta(9,12,15)), respectively, by Delta(5)-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C. reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Delta(5)-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Delta(5)-desaturase activity on 18:1Delta(9) but not 18:1Delta(11). Unexpectedly, CrDES also showed Delta(7)-desaturase activity on 20:2Delta(11,14) and 20:3Delta(11,14,17) to produce 20:3Delta(7,11,14) and 20:4Delta(7,11,14,17), respectively. Since both the Delta(5) bond in C18 and the Delta(7) bond in C20 fatty acids are 'omega13' double bonds, these results indicate that CrDES has omega13 desaturase activity for omega9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.

Amino Acid Sequence↗
PubMed2005

Pexophagy: the selective autophagy of peroxisomes.

Pichia pastoris and Hansenula polymorpha are methylotrophic yeasts capable of utilizing methanol, as a sole source of carbon and energy. Growth of these yeast species on methanol requires the synthesis of cytosolic and peroxisomal enzymes combined with the proliferation of peroxisomes. Peroxisomes are also abundantly present in the alkane-utilizing yeast Yarrowia lipolytica upon growth of cells on oleic acid. This feature has made these yeast species attractive model systems to dissect the molecular mechanisms controlling peroxisome biogenesis. We have found that upon glucose- or ethanol-induced catabolite inactivation, metabolically superfluous peroxisomes are rapidly and selectively degraded within the vacuole by a process called pexophagy, the selective removal of peroxisomes by autophagy-like processes. Utilizing several genetic screens, we have identified a number of genes that are essential for pexophagy. In this review, we will summarize our current knowledge of the molecular events of pexophagy.

Autophagy↗
PubMed2005

Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism.

Methanol is a valuable raw material used in the manufacture of useful chemicals as well as a potential source of energy to replace coal and petroleum. Biotechnological interest in the microbial utilization of methanol has increased because it is an ideal carbon source and can be produced from renewable biomass. Formaldehyde, a cytotoxic compound, is a central metabolic intermediate in methanol metabolism. Therefore, microorganisms utilizing methanol have adopted several metabolic strategies to cope with the toxicity of formaldehyde. Formaldehyde is initially detoxified through trapping by some cofactors, such as glutathione, mycothiol, tetrahydrofolate, and tetrahydromethanopterin, before being oxidized to CO2. Alternatively, free formaldehyde can be trapped by sugar phosphates as the first reaction in the C1 assimilation pathways: the xylulose monophosphate pathway for yeasts and the ribulose monophosphate (RuMP) pathway for bacteria. In yeasts, although formaldehyde generation and consumption takes place in the peroxisome, the cytosolic formaldehyde oxidation pathway also plays a role in formaldehyde detoxification as well as energy formation. The key enzymes of the RuMP pathway are found in a variety of microorganisms including bacteria and archaea. Regulation of the genes encoding these enzymes and their catalytic mechanisms depend on the physiological traits of these organisms during evolution.

Archaea↗
PubMed2005

Cloning and characterization of a cDNA encoding beta-amyrin synthase from petroleum plant Euphorbia tirucalli L.

Euphorbia tirucalli L., known as the petroleum plant, produces a large amount of triterpenes, such as beta-amyrin. Degenerate RT-PCR based on the sequences conserved among known beta-amyrin synthases led to cloning of a putative triterpene synthase cDNA, EtAS, from leaves of E. tirucalli. The deduced amino acid sequence of the EtAS cDNA showed the highest identity of 82% to the Panax ginseng beta-amyrin synthase. Heterologous expression of the EtAS ORF in the methylotrophic yeast, Pichia pastoris, resulted in production of beta-amyrin, revealing that the EtAS cDNA codes for a beta-amyrin synthase. This is the first report of a gene involved in the triterpene synthetic pathway from Euphorbiaceae plants.

Amino Acid Sequence↗
PubMed2005

HxlR, a member of the DUF24 protein family, is a DNA-binding protein that acts as a positive regulator of the formaldehyde-inducible hxlAB operon in Bacillus subtilis.

The HxlR protein from Bacillus subtilis belongs to the DUF24 protein family (InterPro No. IPR002577) of unknown function. The hxlR gene that encodes this protein is located upstream of the hxlAB operon. This operon encodes two key enzymes in the ribulose monophosphate pathway that are involved in formaldehyde fixation, 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. Expression of the hxlAB operon is induced by the presence of formaldehyde. Recombinant HxlR prepared from Escherichia coli showed specific binding to a region of DNA upstream of the hxlAB operon. Using gel-retardation and DNase I footprinting assays, we identified two 25 bp binding regions for HxlR within the upstream DNA. Surface plasmon resonance analyses suggested that two HxlR dimers sequentially bound to the DNA. Finally, we demonstrated that each of the two binding regions for HxlR was necessary for formaldehyde-induced expression of the hxlAB operon in B. subtilis. Thus, we have shown that HxlR is a DNA-binding protein that is necessary for formaldehyde-induced expression of hxlAB in B. subtilis.

Aldehyde-Lyases↗
PubMed2005

The archaeon Pyrococcus horikoshii possesses a bifunctional enzyme for formaldehyde fixation via the ribulose monophosphate pathway.

Pyrococcus horikoshii OT3, a hyperthermophilic and anaerobic archaeon, was found to have an open reading frame (PH1938) whose deduced amino acid sequence of the N-terminal and C-terminal halves showed significant similarity to two key enzymes of the ribulose monophosphate pathway for formaldehyde fixation in methylotrophic bacteria, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), respectively. The organism constitutively produced the encoded protein and exhibited activity of the sequential HPS- and PHI-mediated reactions in a particulate fraction. The full-length gene encoding the hybrid enzyme, the sequence corresponding to the HPS region, and the sequence corresponding to the PHI region were expressed in Escherichia coli and were found to produce active enzymes, rHps-Phi, rHps, or rPhi, respectively. Purified rHps-Phi and rHps were found to be active at the growth temperatures of the parent strain, but purified rPhi exhibited significant susceptibility to heat, suggesting that thermostability of the PHI moiety of the bifunctional enzyme (rHps-Phi) resulted from fusion with HPS. The bifunctional enzyme catalyzed the sequential reaction much more efficiently than a mixture of rHps and rPhi. These and other biochemical characterizations of the PH1938 gene product suggest that the ribulose monophosphate pathway plays a significant role in the archaeon under extreme environmental conditions.

Aldehyde-Lyases↗