Priming and elongation of chitin chains: Implications for chitin synthase mechanism.

Most fungi have multiple chitin synthases (CSs) that may make chitin at different sites on the cell surface, at different times during growth, and in response to cell wall stress. The structure-based model for CS function is for transfer of GlcNAc from UDP-GlcNAc at the cytoplasmic face of the plasma membrane to the non-reducing end of a growing chitin chain, which is concomitantly translocated through a transmembrane channel formed by the synthase. Two aspects of CS mechanism are investigated: how chains might be initiated, and what governs how long they can get.

Identification of pathogenic fungi with an optoelectronic nose.

Human fungal infections have gained recent notoriety following contamination of pharmaceuticals in the compounding process. Such invasive infections are a more serious global problem, especially for immunocompromised patients. While superficial fungal infections are common and generally curable, invasive fungal infections are often life-threatening and much harder to diagnose and treat.

Multitarget drug discovery for tuberculosis and other infectious diseases.

We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogues of the new tuberculosis (TB) drug SQ109 (1), which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters.

Architecture and biosynthesis of the Saccharomyces cerevisiae cell wall.

The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes.

Uptake of radiolabeled GlcNAc into Saccharomyces cerevisiae via native hexose transporters and its in vivo incorporation into GPI precursors in cells expressing heterologous GlcNAc kinase.

Yeast glycan biosynthetic pathways are commonly studied through metabolic incorporation of an exogenous radiolabeled compound into a target glycan. In Saccharomyces cerevisiae glycosylphosphatidylinositol (GPI) biosynthesis, [(3) H]inositol has been widely used to identify intermediates that accumulate in conditional GPI synthesis mutants. However, this approach also labels non-GPI lipid species that overwhelm detection of early GPI intermediates during chromatography.

Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids.

Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring >20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane.

In vivo characterization of the GPI assembly defect in yeast mcd4-174 mutants and bypass of the Mcd4p-dependent step in mcd4Delta cells.

Yeast mcd4-174 mutants are blocked in glycosylphosphatidylinositol (GPI) anchoring of protein, but the stage at which GPI biosynthesis is interrupted in vivo has not been identified, and Mcd4p has also been implicated in phosphatidylserine and ATP transport. We report that the major GPI that accumulates in mcd4-174 in vivo is Man(2)-GlcN-(acyl-Ins)PI, consistent with proposals that Mcd4p adds phosphoethanolamine to the first mannose of yeast GPI precursors. Mcd4p-dependent modification of GPIs can partially be bypassed in the mcd4-174/gpi11 double mutant and in mcd4Delta; mutants by high-level expression of PIG-B and GPI10, which respectively encode the human and yeast mannosyltransferases that add the third mannose of the GPI precursor.

Gpi19, the Saccharomyces cerevisiae homologue of mammalian PIG-P, is a subunit of the initial enzyme for glycosylphosphatidylinositol anchor biosynthesis.

Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-N-acetylglucosaminyltransferase (GPI-GlcNAc transferase).

Saccharomyces cerevisiae Ybr004c and its human homologue are required for addition of the second mannose during glycosylphosphatidylinositol precursor assembly.

Addition of the second mannose is the only obvious step in glycosylphosphatidylinositol (GPI) precursor assembly for which a responsible gene has not been discovered. A bioinformatics-based strategy identified the essential Saccharomyces cerevisiae Ybr004c protein as a candidate for the second GPI alpha-mannosyltransferase (GPI-MT-II). S.

Deficiencies in the essential Smp3 mannosyltransferase block glycosylphosphatidylinositol assembly and lead to defects in growth and cell wall biogenesis in Candida albicans.

Glycosylphosphatidylinositols (GPIs) are essential for viability in yeast and have key roles in cell wall construction. Assembly of Saccharomyces cerevisiae GPIs includes the addition of a fourth, side-branching mannose (Man) to the third Man of the core GPI glycan by the Smp3 mannosyltransferase. The SMP3 gene from the human pathogenic fungus Candida albicans has been cloned.

Human Smp3p adds a fourth mannose to yeast and human glycosylphosphatidylinositol precursors in vivo.

Yeast and human glycosylphosphatidylinositol (GPI) precursors differ in the extent to which a fourth mannose is present as a side branch of the third core mannose. A fourth mannose addition to GPIs has scarcely been detected in studies of mammalian GPI synthesis but is an essential step in the Saccharomyces cerevisiae pathway. We report that human SMP3 encodes a functional homolog of the yeast Smp3 GPI fourth mannosyl-transferase.

Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER.

The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes. Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins.

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E.

The GPI1 homologue from Plasmodium falciparum complements a Saccharomyces cerevisiae GPI1 anchoring mutant.

Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins. The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine to phosphatidylinositol (PI). This chemically simple step is genetically complex because three or four genes are required in both yeast (GPI1, GPI2 and GPI3) and mammals (GPI1, PIG A, PIG H and PIG C), respectively.

Ynl038wp (Gpi15p) is the Saccharomyces cerevisiae homologue of human Pig-Hp and participates in the first step in glycosylphosphatidylinositol assembly.

Glycosylphosphatidylinositols (GPIs) are found in all eukaryotes and are synthesized in a pathway that starts with the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI). This reaction is carried out by a protein complex, three of whose subunits in humans, hGpi1p, Pig-Cp and Pig-Ap, have sequence and functional homologues in the Saccharomyces cerevisiae Gpi1, Gpi2 and Gpi3 proteins, respectively. Human GlcNAc-PI synthase contains two further subunits, Pig-Hp and PigPp.

The essential Smp3 protein is required for addition of the side-branching fourth mannose during assembly of yeast glycosylphosphatidylinositols.

The major glycosylphosphatidylinositols (GPIs) transferred to protein in mammals and trypanosomes contain three mannoses. In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, alpha1,2-linked Man on the alpha1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein. We report that temperature-sensitive smp3 mutants accumulate a GPI containing three mannoses and that smp3 is epistatic to the gpi11, gpi13, and gaa1 mutations, which normally result in the accumulation of Man(4)-GPIs, including the presumed substrate for the yeast GPI transamidase.

Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme.

Dolichol phosphate mannose (DPM) synthase activity, which is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase.

Photoaffinity labelling with P3-(4-azidoanilido)uridine 5'-triphosphate identifies gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis.

Glycosylphosphatidylinositols (GPIs) are made by all eukaryotes. The first step in their synthesis is the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI). Four proteins in mammals and at least three in yeast make up a complex that carries out this reaction.

Glycosylphosphatidylinositol biosynthesis defects in Gpi11p- and Gpi13p-deficient yeast suggest a branched pathway and implicate gpi13p in phosphoethanolamine transfer to the third mannose.

Glycosylphosphatidylinositols (GPIs) are critical for membrane anchoring and intracellular transport of certain secretory proteins. GPIs have a conserved trimannosyl core bearing a phosphoethanolamine (EthN-P) moiety on the third mannose (Man-3) through which the glycolipid is linked to protein, but diverse GPI precursors with EthN-Ps on Man-1 and Man-2 have also been described. We report on two essential yeast genes whose products are required late in GPI assembly.

Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes.

Membrane anchoring of cell surface proteins via glycosylphosphatidylinositol (GPI) occurs in all eukaryotic organisms. In addition, GPI-related glycophospholipids are important constituents of the glycan coat of certain protozoa. Defects in GPI biosynthesis can retard, if not abolish growth of these organisms.

MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast.

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway.