Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Abstract

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...

Atg26 is Not Involved in Autophagy-Related Pathways inSaccharomyces cerevisiae

“…A truncated form of the vacuolar alkaline phosphatase (Pho8), Pho8∆60, can only be transported to the vacuole through autophagy. 13 Once delivered to the vacuole, it is proteolytically processed to remove a C-terminal propeptide, and acquires its phosphatase activity. 7 Thus, we can quantitatively measure autophagic activity by monitoring Pho8∆60-dependent alkaline and atg26∆ cells were grown in rich medium to mid-log phase, and then shifted to starvation medium for four hours.…”

“…It seems that the role of PpAtg26 in pexophagy is unique to P. pastoris, although the C-terminal UDP-glucosyl transferase domain has very high sequence identity between these two yeasts. It could be that the amount of sterol glucoside (SG) in S. cerevisiae 13 is too low to have any effect on these autophagy-related pathways. SG synthesis is higher in P. pastoris, and the SG content might influence MIPA formation and other membrane events in P. pastoris, which contributes to the involvement of PpAtg26 in pexophagy; 5 however, the role of the S. cerevisiae Atg26 protein, remains to be elucidated.…”

Novel Predicted Peptidases with a Potential Role in the Ubiquitin Signaling Pathway

“…Enzymes of this superfamily are typically involved in modification of peptides and small molecules including sugars, steroids, phenols and terpenoid alcohols. 44 The glycosyltransferases of CHV3 and CHV4 are closely related to fungal steroid glycosyltransferases which are involved in the modification of lipid head groups. 44 This suggests that the glycosyltransferase and the PPPDE peptidase are recent HGTs from fungi to the ancestor of the CHV3 and CHV4 hypoviruses, and that PPPDE might have partially or completely displaced the ancestral HC-Pro-like proteases in these viruses.…”

“…44 The glycosyltransferases of CHV3 and CHV4 are closely related to fungal steroid glycosyltransferases which are involved in the modification of lipid head groups. 44 This suggests that the glycosyltransferase and the PPPDE peptidase are recent HGTs from fungi to the ancestor of the CHV3 and CHV4 hypoviruses, and that PPPDE might have partially or completely displaced the ancestral HC-Pro-like proteases in these viruses. The viral PPPDE family consists of highly divergent members of the superfamily, namely the VP2 protein of plant oryzaviruses and insect cypoviruses (members of the reovirus family), and the minor structural protein of the parvo-like ssDNA virus, Bombyx mori densonucleosis virus type 2 (BmDV2).…”

Characterization of Sterol Lipids in Kluyveromyces lactis Strain M-16 Accumulating a High Amount of Steryl Glucoside

“…Steryl glucoside (SG), which is composed of the sterol ring with a glucose residue at the C 3 -OH group, is included in the membrane lipids of various organisms 1,2) . In plants, SG may serve as a primer for cellulose biosynthesis because the cotton fiber membrane synthesizes sitosterolcellodextrins from SG and UDP-glucose 3) .…”