Species-Specific Differences in the Susceptibility of Fungi to the Antifungal Protein AFP Depend on C-3 Saturation of Glycosylceramides.

AFP is an antimicrobial peptide (AMP) produced by the filamentous fungus Aspergillus giganteus and is a very potent inhibitor of fungal growth that does not affect the viability of bacteria, plant, or mammalian cells. It targets chitin synthesis and causes plasma membrane permeabilization in many human- and plant-pathogenic fungi, but its exact mode of action is not known. After adoption of the "damage-response framework of microbial pathogenesis" regarding the analysis of interactions between AMPs and microorganisms, we have recently proposed that the cytotoxic capacity of a given AMP depends not only on the presence/absence of its target(s) in the host and the AMP concentration applied but also on other variables, such as microbial survival strategies. We show here using the examples of three filamentous fungi (Aspergillus niger, Aspergillus fumigatus, and Fusarium graminearum) and two yeasts (Saccharomyces cerevisiae and Pichia pastoris) that the important parameters defining the AFP susceptibilities of these fungi are (i) the presence/absence of glycosylceramides, (ii) the presence/absence of Δ3(E) desaturation of the fatty acid chain therein, and (iii) the (dis)ability of these fungi to respond to AFP inhibitory effects with the fortification of their cell walls via increased chitin and ß-(1,3)-glucan synthesis. These observations support the idea of the adoption of the damage-response framework to holistically understand the outcome of AFP inhibitory effects.IMPORTANCE Our data suggest a fundamental role of glycosylceramides in the susceptibility of fungi to AFP. We discovered that only a minor structural difference in these molecules-namely, the saturation level of their fatty acid chain, controlled by a 2-hydroxy fatty N-acyl-Δ3(E)-desaturase-represents a key to understanding the inhibitory activity of AFP. As glycosylceramides are important components of fungal plasma membranes, we propose a model which links AFP-mediated inhibition of chitin synthesis in fungi with its potential to disturb plasma membrane integrity.

Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris.

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.

Atg35, a micropexophagy-specific protein that regulates micropexophagic apparatus formation in Pichia pastoris.

Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAS), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. However, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35∆ mutant was not affected in macropexophagy, cytoplasm-to-vacuole targeting or general autophagy. However, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MIPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral ER to the nuclear envelope was required to organize a peri-nuclear structure (PNS), the site of Atg35 colocalization during micropexophagy. In summary, Atg35 is a new Atg protein that relocates to the PNS and specifically regulates MIPA formation during micropexophagy.

Biosynthesis of sphingolipids in plants (and some of their functions).

Our knowledge of plant sphingolipid metabolism and function has significantly increased over the past years. This applies mainly to the identification and the functional characterization of genes and enzymes involved in sphingolipid biosynthesis. In addition a number of plant mutants have provided new insights into sphingolipid functions. Very little is still known about intracellular transport, spatial distribution, degradation and signaling functions of sphingolipids. However, combination of Arabidopsis genetics with lipidomics and cell biology will soon bring our understanding of these issues to a new level.

The functions of steryl glycosides come to those who wait: Recent advances in plants, fungi, bacteria and animals.

The attachment of a sugar moiety to the 3-hydroxy group of a sterol drastically increases the size of the hydrophilic part of the lipid. It is obvious that the glycosylation of a considerable fraction of membrane-bound free sterols alters the biophysical properties of the membrane. However, the consequences of such changes in the proportions of free sterols and steryl glycosides on the biological functions of a membrane are still unclear. This is the main hurdle in understanding the biological functions of steryl glycosides on a molecular level. The recent cloning of sterol glycosyltransferase genes from plants, fungi and bacteria has enabled genetic approaches to analyze steryl glycoside functions. Down regulation of phytosteryl beta-glycoside biosynthesis in Arabidopsis thaliana causes several dysfunctions in seed development. Ergosteryl beta-glycoside depleted mutants of the yeast Pichia pastoris lose their ability to degrade their peroxisomes by an autophagic mechanism called micropexophagy. In the plant-pathogenic fungus Colletotrichum orbiculare the same defect impairs invasion of the cucumber host plants. Helicobacter pylori, a bacterium colonizing the human stomach, is unable to modulate the host's immune response when the cholesteryl alpha-glycoside biosynthesis of the bacterium is mutated. These mutants with manipulated steryl glycoside metabolism will inspire further studies with cell biological, biophysical and other methods that will provide us with a mechanistic understanding of steryl glycoside functions.

Glycolipid headgroup replacement: a new approach for the analysis of specific functions of glycolipids in vivo.

Glycolipids with one or two sugar residues attached to different lipid backbones are found in biomembranes of bacteria, fungi, plants and animals in the form of steryl glycosides, glycosylceramides and diacylglycerol glycosides. They contain different sugar residues, mainly glucose and galactose, in either alpha- or beta-configuration. Many of the isolated compounds have been studied in great detail with regard to their biophysical behavior in artificial membrane systems. With the availability of cloned genes, the methods of reverse genetics were used to study glycolipid functions in living cells. The deletion of a lipid glycosyltransferase gene leads to the loss of the corresponding glycolipid in the transformed pro- and eukaryotic organisms. Often, these glycosyltransferase deletion mutants showed many differences to the wild-type organisms and thus demonstrated the biological importance of the glycolipid. When extensive deletion-induced glycolipid losses were not complemented by higher proportions of other membrane lipids, the mutants could display severe phenotypes due to a serious dysfunction or even collapse of an entire membrane system. On the other hand, by this approach the specific contribution of characteristic head group details cannot be recognized and separated from more general glycolipid functions. Many of these difficulties can be circumvented by a glycolipid headgroup replacement approach. This new approach requires the exchange of a lipid glycosyltransferase in an organism by a heterologous glycosyltransferase having a different headgroup specificity, e.g. the substitution of a galactosyltransferase by a glucosyltransferase. The resulting transgenic organism produces a novel glycolipid which differs from that of the native organism not in proportion, but only in structural details of its headgroup. Therefore, such rescued mutants are comparable to suppressor mutants and show less severe phenotypes than the intermediate deletion mutants. A comparison between the wild type, the simple deletion mutant and the mutant rescued by glycolipid replacement will not only disclose general functions of glycolipids, but also additional roles of headgroup details.

Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1-phosphate in Arabidopsis.

The role of Delta4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Delta4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Delta4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Delta4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Delta4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Delta4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Delta4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Delta4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides.

Identification and functional characterization of the 2-hydroxy fatty N-acyl-Delta3(E)-desaturase from Fusarium graminearum.

Delta3(E)-unsaturated fatty acids are characteristic components of glycosylceramides from some fungi, including also human- and plant-pathogenic species. The function and genetic basis for this unsaturation is unknown. For Fusarium graminearum, which is pathogenic to grasses and cereals, we could show that the level of Delta3-unsaturation of glucosylceramide (GlcCer) was highest at low temperatures and decreased when the fungus was grown above 28 degrees C. With a bioinformatics approach, we identified a new family of polypeptides carrying the histidine box motifs characteristic for membrane-bound desaturases. One of the corresponding genes was functionally characterized as a sphingolipid-Delta3(E)-desaturase. Deletion of the candidate gene in F. graminearum resulted in loss of the Delta3(E)-double bond in the fatty acyl moiety of GlcCer. Heterologous expression of the corresponding cDNA from F. graminearum in the yeast Pichia pastoris led to the formation of Delta3(E)-unsaturated GlcCer.

Cholesterol glucosylation promotes immune evasion by Helicobacter pylori.

Helicobacter pylori infection causes gastric pathology such as ulcer and carcinoma. Because H. pylori is auxotrophic for cholesterol, we have explored the assimilation of cholesterol by H. pylori in infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glucosylation. Excessive cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells, such as macrophages and dendritic cells, and enhances antigen-specific T cell responses. A cholesterol-rich diet during bacterial challenge leads to T cell-dependent reduction of the H. pylori burden in the stomach. Intrinsic alpha-glucosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. We identify the gene hp0421 as encoding the enzyme cholesterol-alpha-glucosyltransferase responsible for cholesterol glucosylation. Generation of knockout mutants lacking hp0421 corroborates the importance of cholesteryl glucosides for escaping phagocytosis, T cell activation and bacterial clearance in vivo. Thus, we propose a mechanism regulating the host-pathogen interaction whereby glucosylation of a lipid tips the scales towards immune evasion or response.

Cloning of a cholesterol-alpha-glucosyltransferase from Helicobacter pylori.

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase.

Functional differences between galactolipids and glucolipids revealed in photosynthesis of higher plants.

Galactolipids represent the most abundant lipid class in thylakoid membranes, where oxygenic photosynthesis is performed. The identification of galactolipids at specific sites within photosynthetic complexes by x-ray crystallography implies specific roles for galactolipids during photosynthetic electron transport. The preference for galactose and not for the more abundant sugar glucose in thylakoid lipids and their specific roles in photosynthesis are not understood. Introduction of a bacterial glucosyltransferase from Chloroflexus aurantiacus into the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana resulted in the accumulation of a glucose-containing lipid in the thylakoids. At the same time, the growth defect of the dgd1 mutant was complemented. However, the degree of trimerization of light-harvesting complex II and the photosynthetic quantum yield of transformed dgd1 plants were only partially restored. These results indicate that specific interactions of the galactolipid head group with photosynthetic protein complexes might explain the preference for galactose in thylakoid lipids of higher plants. Therefore, galactose in thylakoid lipids can be exchanged with glucose without severe effects on growth, but the presence of galactose is crucial to maintain maximal photosynthetic efficiency.

Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling.

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Delta4,8-desaturated ceramide as substrates.

An inhibitor of glucosylceramide synthase inhibits the human enzyme, but not enzymes from other organisms.

Specific inhibitors of glucosylceramide biosynthesis are used as drugs for the treatment of some human diseases correlated to glycosphingolipid metabolism. The target of the presently available inhibitors is the human glucosylceramide synthase (GCS), but effects on enzymes from other organisms have not been studied. We expressed cDNAs encoding GCS enzymes from lower animals, plants, fungi, and bacteria in the yeast P. pastoris. In vitro GCS assays with the GCS inhibitor D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol showed that this inhibitor did not affect non-human GCS enzymes.

Glycoengineering of cyanobacterial thylakoid membranes for future studies on the role of glycolipids in photosynthesis.

The lipid composition of thylakoid membranes is conserved from cyanobacteria to angiosperms. The predominating components are monogalactosyl- and digalactosyldiacylglycerol. In cyanobacteria, thylakoid membrane biosynthesis starts with the formation of monoglucosyldiacylglycerol which is C4-epimerized to the corresponding galactolipid, whereas in plastids monogalactosyldiacylglycerol is formed at the beginning. This suggests that galactolipids have specific functions in thylakoids. We wanted to investigate whether galactolipids can be replaced by glycosyldiacylglycerols with headgroups differing in their epimeric and anomeric details as well as the attachment point of the terminal hexose in diglycosyldiacylglycerols. For this purpose putative glycosyltransferase sequences were identified in databases to be used for functional expression in various host organisms. From 18 newly identified sequences, four turned out to encode glycosyltransferases catalyzing final steps in glycolipid biosynthesis: two alpha-glucosyltransferases, one beta-galactosyltransferase and one beta-glucosyltransferase. Their functional annotation was based on detailed structural characterization of the new glycolipids formed in the transformant hosts as well as on in vitro enzymatic assays. The expression of alpha-glucosyltransferases in the cyanobacterium Synechococcus resulted in the accumulation of the new alpha-galactosyldiacylglycerol which is ascribed to epimerization of the corresponding glucolipid. The expression of the beta-glucosyltransferase led to a high proportion of new beta-glucosyl-(1-->6)-beta-galactosyldiacylglycerol almost entirely replacing the native digalactosyldiacylglycerol. These results demonstrate that modifications of the glycolipid pattern in thylakoids are possible.

Processive lipid galactosyl/glucosyltransferases from Agrobacterium tumefaciens and Mesorhizobium loti display multiple specificities.

The glycosyltransferase family 21 (GT21) includes both enzymes of eukaryotic and prokaryotic organisms. Many of the eukaryotic enzymes from animal, plant, and fungal origin have been characterized as uridine diphosphoglucose (UDP-Glc):ceramide glucosyltransferases (glucosylceramide synthases [Gcs], EC 2.4.1.80). As the acceptor molecule ceramide is not present in most bacteria, the enzymatic specificities and functions of the corresponding bacterial glycosyltransferases remain elusive. In this study, we investigated the homologous and heterologous expression of GT21 enzymes from Agrobacterium tumefaciens and Mesorhizobium loti in A. tumefaciens, Escherichia coli, and the yeast Pichia pastoris. Glycolipid analyses of the transgenic organisms revealed that the bacterial glycosyltransferases are involved in the synthesis of mono-, di- and even tri-glycosylated glycolipids. As products resulting from their activity, we identified 1,2-diacyl-3-(O-beta-D-galacto-pyranosyl)-sn-glycerol, 1,2-diacyl-3-(O-beta-D-gluco-pyranosyl)-sn-glycerol as well as higher glycosylated lipids such as 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-gluco-pyranosyl]-sn-glycerol, and the deviatingly linked diglycosyldiacylglycerol 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->3)-O-beta-D-galacto-pyranosyl]-sn-glycerol. From a mixture of triglycosyldiacylglycerols, 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol could be separated in a pure form. In vitro enzyme assays showed that the glycosyltransferase from A. tumefaciens favours uridine diphosphogalactose (UDP-Gal) over UDP-Glc. In conclusion, the bacterial GT21 enzymes differ from the eukaryotic ceramide glucosyltransferases by the successive transfer of up to three galactosyl and glucosyl moieties to diacylglycerol.

Analysis of the vitamin B6 biosynthesis pathway in the human malaria parasite Plasmodium falciparum.

Vitamin B6 is an essential cofactor for more than 100 enzymatic reactions. Mammalian cells are unable to synthesize vitamin B6 de novo, whereas bacteria, plants, fungi, and as shown here Plasmodium falciparum possess a functional vitamin B6 synthesis pathway. P. falciparum expresses the proteins Pdx1 and Pdx2, corresponding to the yeast enzymes Snz1-p and Sno1-p, which are essential for the vitamin B6 biosynthesis. An involvement of PfPdx1 and PfPdx2 in the de novo synthesis of vitamin B6 was shown by complementation of pyridoxine auxotroph yeast cells. Both plasmodial proteins act together in the glutaminase activity with a specific activity of 209 nmol min(-1) mg(-1) and a K(m) value for glutamine of 1.3 mm. Incubation of the parasites with methylene blue revealed by Northern blot analysis an elevated transcriptional level of pdx1 and pdx2, suggesting a participation of these proteins in the defenses against singlet oxygen. To be an active cofactor, vitamin B6 has to be phosphorylated by the pyridoxine kinase (PdxK). The recombinant plasmodial PdxK revealed K(m) values for the B6 vitamers pyridoxine and pyridoxal and for ATP of 212, 70, and 82 microM, respectively. All three enzymes expose a stage-specific transcription pattern within the trophozoite stage that guarantees the concurrent expression of Pdx1, Pdx2, and PdxK for the indispensable provision of vitamin B6. The occurrence of the vitamin B6 de novo synthesis pathway displays a potential new drug target, which can be exploited for the development of new chemotherapeutics against the human malaria parasite P. falciparum.

Defensins from insects and plants interact with fungal glucosylceramides.

Growth of the yeast species Candida albicans and Pichia pastoris is inhibited by RsAFP2, a plant defensin isolated from radish seed (Raphanus sativus), at micromolar concentrations. In contrast, gcs-deletion mutants of both yeast species are resistant toward RsAFP2. GCS genes encode UDP-glucose:ceramide glucosyltransferases, which catalyze the final step in the biosynthesis of the membrane lipid glucosylceramide. In an enzyme-linked immunosorbent assay-based binding assay, RsAFP2 was found to interact with glucosylceramides isolated from P. pastoris but not with soybean nor human glucosylceramides. Furthermore, the P. pastoris parental strain is sensitive toward RsAFP2-induced membrane permeabilization, whereas the corresponding gcs-deletion mutant is highly resistant to RsAFP2-mediated membrane permeabilization. A model for the mode of action of RsAFP2 is presented in which all of these findings are linked. Similarly to RsAFP2, heliomicin, a defensin-like peptide from the insect Heliothis virescens, is active on C. albicans and P. pastoris parental strains but displays no activity on the gcs-deletion mutants of both yeast species. Furthermore, heliomicin interacts with glucosylceramides isolated from P. pastoris and soybean but not with human glucosylceramides. These data indicate that structurally homologous anti-fungal peptides present in species from different eukaryotic kingdoms interact with the same target in the fungal plasma membrane, namely glucosylceramides, and as such support the hypothesis that defensins from plants and insects have evolved from a single precursor.

Formation of glucosylceramide and sterol glucoside by a UDP-glucose-dependent glucosylceramide synthase from cotton expressed in Pichia pastoris.

In plants, glucosylceramide (GlcCer) biosynthesis is poorly understood. Previous investigations suggested that sterol glucoside (SG) acts as the actual glucose donor for the plant GlcCer synthase (GCS). We addressed this question by generating a Pichia pastoris double mutant devoid of GlcCer and SG. This mutant was used for heterologous expression of the plant GCS. The activity of the GCS resulted in the accumulation of GlcCer and, surprisingly, a small proportion of SG. The synthesis of GlcCer in the transformed double mutant shows that the GCS is SG-independent, while the detection of SG suggests that in addition to the sterol glucosyltransferase, also the GCS may contribute in planta to SG biosynthesis.

Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica.

Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.

Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domain.

Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.