Physiological role of the FDH1 gene in methylotrophic yeast Komagataella phaffii and carbon recovery as formate during methylotrophic growth of the fdh1-deletion strain.

During methylotrophic growth of Komagataella phaffii, a large amount of carbon is lost as CO. In this study, we aimed to construct a recovery system for carbon atoms, which emit as CO along the methanol dissimilation pathway in the form of formate when using strain fdh1Δ, the deletion mutant of formate dehydrogenase gene (FDH1). Strain fdh1Δ showed a severe growth defect when using methanol as the sole carbon source.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression.

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression.

Regulation of alcohol oxidase gene expression in methylotrophic yeast Ogataea minuta.

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta possesses strongly methanol-inducible genes, such as AOX1.

Yeast Dop1 is required for glycosyltransferase retrieval from the trans-Golgi network.

Background: Glycosyltransferases are type II membrane proteins that are responsible for glycan modification of proteins and lipids, and localize to distinct cisternae in the Golgi apparatus. During cisternal maturation, retrograde trafficking helps maintain the steady-state localization of these enzymes in the sub-compartments of the Golgi.

Methods: To understand how glycosyltransferases are recycled in the late Golgi complex, we searched for genes that are essential for budding yeast cell growth and that encode proteins localized in endosomes and in the Golgi.

Lipid moiety of glycosylphosphatidylinositol-anchored proteins contributes to the determination of their final destination in yeast.

Yeasts have two classes of glycosylphosphatidylinositol (GPI)-anchored proteins; one is transferred to the cell wall, whereas the other is retained on the plasma membrane. The lipid moieties of the GPI in Saccharomyces cerevisiae consist of either phosphatidylinositol (PI) or inositolphosphorylceramide (IPC). Cwh43p is involved in the remodeling of lipid from PI to IPC.

Mating type switching, formation of diploids, and sporulation in the methylotrophic yeast Ogataea minuta.

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta also possesses strongly methanol-inducible genes, such as AOX1.

Structural and functional analysis of Alg1 beta-1,4 mannosyltransferase reveals the physiological importance of its membrane topology.

In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a β-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate.

PER1, GUP1 and CWH43 of methylotrophic yeast Ogataea minuta are involved in cell wall integrity.

In eukaryotes, the glycosylphosphatidylinositol (GPI) modification of many glycoproteins on the cell surface is highly conserved. The lipid moieties of GPI-anchored proteins undergo remodelling processes during their maturation. To date, the products of the PER1, GUP1 and CWH43 genes of the yeast Saccharomyces cerevisiae have been shown to be involved in the lipid remodelling.

Identification of Novel Peptidyl Serine α-Galactosyltransferase Gene Family in Plants.

In plants, serine residues in extensin, a cell wall protein, are glycosylated with O-linked galactose. However, the enzyme that is involved in the galactosylation of serine had not yet been identified. To identify the peptidyl serine O-α-galactosyltransferase (SGT), we chose Chlamydomonas reinhardtii as a model.

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast.

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI-anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A2 Per1p and O-acyltransferase Gup1p play essential roles in diacylglycerol-type lipid remodelling of GPI-anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide.

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p.

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively.

Characterization of endoplasmic reticulum-localized UDP-D-galactose: hydroxyproline O-galactosyltransferase using synthetic peptide substrates in Arabidopsis.

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses.

O-mannosylation is required for degradation of the endoplasmic reticulum-associated degradation substrate Gas1*p via the ubiquitin/proteasome pathway in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, protein O-mannosylation, which is executed by protein O-mannosyltransferases, is essential for a variety of biological processes as well as for conferring solubility to misfolded proteins. To determine if O-mannosylation plays an essential role in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, we used a model misfolded protein, Gas1*p. The O-mannose content of Gas1*p, which is transferred by protein O-mannosyltransferases, was higher than that of Gas1p.

The cytoplasmic region of alpha-1,6-mannosyltransferase Mnn9p is crucial for retrograde transport from the Golgi apparatus to the endoplasmic reticulum in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Och1p and Mnn9p mannosyltransferases are localized in the cis-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always colocalized to the same cisternae.

Saccharomyces cerevisiae CWH43 is involved in the remodeling of the lipid moiety of GPI anchors to ceramides.

The glycosylphosphatidylinositol (GPI)-anchored proteins are subjected to lipid remodeling during their biosynthesis. In the yeast Saccharomyces cerevisiae, the mature GPI-anchored proteins contain mainly ceramide or diacylglycerol with a saturated long-fatty acid, whereas conventional phosphatidylinositol (PI) used for GPI biosynthesis contains an unsaturated fatty acid. Here, we report that S.

Fatty acid remodeling of GPI-anchored proteins is required for their raft association.

Whereas most of the cellular phosphatidylinositol (PI) contain unsaturated fatty chains and are excluded from rafts, GPI-anchored proteins (APs) unusually contain two saturated fatty chains in their PI moiety, and they are typically found within lipid rafts. However, the origin of the saturated chains and whether they are essential for raft association are unclear. Here, we report that GPI-APs, with two saturated fatty chains, are generated from those bearing an unsaturated chain by fatty acid remodeling that occurs most likely in the Golgi and requires post-GPI-attachment to proteins (PGAP)2 and PGAP3.

PER1 is required for GPI-phospholipase A2 activity and involved in lipid remodeling of GPI-anchored proteins.

Glycosylphoshatidylinositol (GPI) anchors are remodeled during their transport to the cell surface. Newly synthesized proteins are transferred to a GPI anchor, consisting of diacylglycerol with conventional C16 and C18 fatty acids, whereas the lipid moiety in mature GPI-anchored proteins is exchanged to either diacylglycerol containing a C26:0 fatty acid in the sn-2 position or ceramide in Saccharomyces cerevisiae. Here, we report on PER1, a gene encoding a protein that is required for the GPI remodeling pathway.

Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p.

In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway.

Inositol deacylation by Bst1p is required for the quality control of glycosylphosphatidylinositol-anchored proteins.

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins.

Comparison of cell wall localization among Pir family proteins and functional dissection of the region required for cell wall binding and bud scar recruitment of Pir1p.

We examined the localization of the Pir protein family (Pir1 to Pir4), which is covalently linked to the cell wall in an unknown manner. In contrast to the other Pir proteins, a fusion of Pir1p and monomeric red fluorescent protein distributed in clusters in pir1Delta cells throughout the period of cultivation, indicating that Pir1p is localized in bud scars. Further microscopic analysis revealed that Pir1p is expressed inside the chitin rings of the bud scars.

GPI7 involved in glycosylphosphatidylinositol biosynthesis is essential for yeast cell separation.

GPI7 is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. We isolated gpi7 mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature. WSC1, RHO2, ROM2, GFA1, and CDC5 genes were isolated as multicopy suppressors of gpi7-2 mutant.

Salt stress induces the expression of Schizosaccharomyces pombe och1+, which encodes an initiation-specific alpha-1,6-mannosyltransferase for N-linked outer chain synthesis of cell wall mannoproteins.

The Schizosaccharomyces pombe Och1p is required for the initiation of outer chain elongation of N-linked oligosaccharides. In this report, we investigated the transcriptional control of the S. pombe och1+ gene and found that the expression of the och1+ gene was not regulated during the cell cycle, but was induced by NaCl and KCl through a transcription factor, Atf1p.

An N-acetylglucosaminyltransferase of the Golgi apparatus of the yeast Saccharomyces cerevisiae that can modify N-linked glycans.

The yeast Saccharomyces cerevisiae is widely regarded as being only capable of producing N-linked glycans with high-mannose structures. To investigate the glycan structures made in different mutant strains, we made use of a reporter protein consisting of a version of hen egg lysozyme that contains a single site for N-linked glycosylation. Mass spectrometry analysis of the attached glycans revealed that a large proportion contained an unexpected extra mass corresponding to a single N-acetylhexosamine residue.