Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose: sterol glucosyltransferase (EC 2.4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 ‫؍‬ YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-␤-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-␤-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.Sterol glycosides are widespread membrane lipids, occurring in all plants, several algae (1-3), some fungi (4 -9), slime molds (10 -12), Dictyostelium (13), a few bacteria (14 -19), and even animals (20 -23). The knowledge base for sterol glycosides is rather limited compared with free sterols and sterol esters, where the synthesis, transport, and functions have been studied extensively in animals (24 -29), plants, (30 -35), and yeast (36 -40). The basis for studies on the functions of sterol glycosides is the assumption that free sterols and sterol glycosides differ physiologically. It is obvious that the attachment of a glycosyl moiety to the sterol backbone alters the physical properties of this lipid. As a result, there are changes in the properties of membranes containing different proportions of free sterols and sterol glycosides. Such changes have been studied with artificial membranes in terms of membrane fluidity, permeability, hydration, and phase behavior (41)(42)(43)(44). However, we still do not know how free sterols and sterol glycosides differ physiologically in biological membranes and why many eukaryotic organisms synthesize sterol glycosides. One of the main reasons for our limited knowledge in this field is the lack of a genetic approach. The objective of the present work was the isolation and characterization of sterol glycosyltransferase genes from eukaryotic organisms. We expect that genetic manipulation of these genes will facilitate the elucidation of sterol glycoside functions in these organisms.The predominating sugar moiety in sterol glycosides is glucose. Besides plants...

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 traf®c and pathogenesis, but its signi®c-ance 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 identi®ed 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.

These authors contributed equally to this study. SummaryGenetic dissection of the lipid bilayer composition provides essential in vivo evidence for the role of individual lipid species in membrane function. To understand the in vivo role of the anionic phospholipid, phosphatidylglycerol, the loss-of-function mutation was identified and characterized in the Arabidopsis thaliana gene coding for phosphatidylglycerophosphate synthase 1, PGP1. This mutation resulted in pigment-deficient plants of the xantha type in which the biogenesis of thylakoid membranes was severely compromised. The PGP1 gene coded for a precursor polypeptide that was targeted in vivo to both plastids and mitochondria. The activity of the plastidial PGP1 isoform was essential for the biosynthesis of phosphatidylglycerol in chloroplasts, whereas the mitochondrial PGP1 isoform was redundant for the accumulation of phosphatidylglycerol and its derivative cardiolipin in plant mitochondrial membranes. Together with findings in cyanobacteria, these data demonstrated that anionic phospholipids play an important, evolutionarily conserved role in the biogenesis and function of the photosynthetic machinery. In addition, mutant analysis suggested that in higher plants, mitochondria, unlike plastids, could import phosphatidylglycerol from the endoplasmic reticulum.

Induction of HSP70 heat shock genes by light has been demonstrated in Chlamydomonas. Our aim was to establish whether this induction by light is mediated by the heat stress sensing pathway or by an independent signal chain. Inhibitors of cytoplasmic protein synthesis revealed an initial difference. Cycloheximide and other inhibitors of protein synthesis prevented HSP70A induction upon illumination but not during heat stress. Analysis of HSP70A induction in cells that had differentiated into gametes revealed a second difference. While heat shock resulted in elevated HSP70A mRNA levels, light was no longer able to serve as an inducer in gametes. To identify the regulatory sequences that mediate the response of the HSP70A gene to either heat stress or light we introduced a series of progressive 5' truncations into its promoter sequence. Analyses of the levels of mRNA transcribed from these deletion constructs showed that in most of them the responses to heat shock and light were similar, suggesting that light induction is mediated by a light-activated heat shock factor. However, we show that the HSP70A promoter also contains cis-acting sequences involved in light induction that do not participate in induction by heat stress. Together, these results provide evidence for a regulation of HSP70A gene expression by light through a heat shock-independent signal pathway.

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10Digital fabrication represents innovative, computer-controlled processes and technologies with the 11 potential to expand the boundaries of conventional construction. Their use in construction is currently 12 restricted to complex and iconic structures, but the growth potential is large. This paper aims to 13 investigate the environmental opportunities of digital fabrication methods, particularly when applied to 14 complex concrete geometries. A case study of a novel robotic additive process that is applied to a wall 15 structure is evaluated with the Life Cycle Assessment (LCA) method. The results of the assessment 16 demonstrate that digital fabrication provides environmental benefits when applied to complex 17 structures. The results also confirm that additional complexity is achieved through digital fabrication 18 without additional environmental costs. This study provides a quantitative argument to position digital 19 fabrication at the beginning of a new era, which is often called the Digital Age in many other 20 disciplines. 21

Two Arabidopsis thaliana genes were shown to encode phosphatidylglycerophosphate synthases (PGPS) of 25.4 and 32.2 kDa, respectively. Apart from their N-terminal regions, the two proteins exhibit high sequence similarity. Functional expression studies in yeast provided evidence that the 25.4 kDa protein is a microsomal PGPS while the 32.2 kDa protein represents a preprotein which can be imported into yeast mitochondria and processed to a mature PGPS. The two isozymes were solubilized and purified as fusion proteins carrying a His tag at their C-terminus. Enzyme assays with both membrane fractions and purified enzyme fractions revealed that the two A. thaliana isozymes have similar properties but differ in their CDP-diacylglycerol species specificity. ß