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

Abstract

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. In this study, we show that despite lacking the ability to metabolize N-acetylglucosamine (GlcNAc), S. cerevisiae is capable of importing low levels of extracellular GlcNAc via almost all members of the hexose transporter family. Furthermore, expression of a heterologous GlcNAc kinase gene permits efficient incorporation of exogenous [(14) C]GlcNAc into nascent GPI structures in vivo, dramatically lowering the background signal from non-GPI lipids. Utilizing this new method with several conditional GPI biosynthesis mutants, we observed and characterized novel accumulating lipids that were not previously visible using [(3) H]inositol labeling. Chemical and enzymatic treatments of these lipids indicated that each is a GPI intermediate likely having one to three mannoses and lacking ethanolamine phosphate (Etn-P) side-branches. Our data support a model of yeast GPI synthesis that bifurcates after the addition of the first mannose and that includes a novel branch that produces GPI species lacking Etn-P side-branches.

Fig 1

Yeast GlcNAc metabolism. Candida albicans imports and metabolizes GlcNAc via a salvage pathway (red text and arrows) that is absent from Saccharomyces cerevisiae. This pathway consists of a GlcNAc-specific transporter (CaNgt1p), GlcNAc kinase (CaNag5p), GlcNAc6P deactylase (CaNag2p), and GlcN6P deaminase (CaNag1p). Saccharomyces cerevisiae cannot metabolize GlcNAc, and instead essential UDP-GlcNAc is formed de novo from Fru6P (black text and arrows), an intermediate in the metabolism of various hexose sugars (e.g., Glc, glucose; Gal, galactose; Man, mannose). Hexoses are internalized by S. cerevisiae cells via several plasma membrane hexose transporters (the Hxt family and Gal2p).

Fig 2

Growth of engineered Saccharomyces cerevisiae strains on GlcNAc. (a) Saccharomyces cerevisiae strains S1–S9 expressing different combinations of Candida albicans GlcNAc salvage pathway genes (NGT1, NAG5, NAG2, and NAG1) were propagated on synthetic agar medium containing 2% glucose or 2% GlcNAc. Patches represent 10-fold serial dilutions of cells prepared as described in the Materials and methods section. (b) The culture density of wt (S1), S8 and S9 strains in liquid synthetic medium containing 2% glucose or 2% GlcNAc measured over 100 h of shaking at 30 °C.

Fig 3

Internalization of [³H]GlcNAc by Saccharomyces cerevisiae cells. Import of extracellular [³H]GlcNAc into the cytoplasm was compared between wild-type cells, cells genetically deleted of all hexose transporter genes (strain VW1000), and VW1000 cells over-expressing individual hexose transporter genes (HXT genes and GAL2). HXT6, HXT11, HXT17, and HXT16 were not included in the study as they are nearly identical to HXT7, HXT9, HXT13, and HXT15, respectively. HXT12 is a pseudogene.

Fig 4

Incorporation of [¹⁴C]GlcNAc into Saccharomyces cerevisiae polysaccharides. (a) Saccharomyces cerevisiae strains expressing different combinations of Candida albicans GlcNAc salvage pathway genes were exposed to extracellular [¹⁴C]GlcNAc. Cell wall chitin was isolated from each strain, and the amount of [¹⁴C]GlcNAc incorporated into chitin was measured by scintillation counting. (b) A fluorograph showing the TLC separation of [¹⁴C]GlcNAc or [³H]inositol-labeled lipids isolated from smp3 cells or (c) wild-type cells expressing various combinations of CaNAG5 and CaNGT1. Solvent B was used for separation (see Materials and methods). The position of a characterized Man₃(Etn-P)-GPI lipid that accumulates in smp3 cells is indicated (Grimme et al., 2001). M, mannose; E, ethanolamine phosphate; G, glucosamine; PI, phosphatidylinositol, Ins, inositol.

Fig 5

Accumulation of an un-substituted Man₁-GPI in gpi18 and mcd4 mutants. (a,b) Conditional gpi18 and mcd4 mutants expressing CaNAG5 were metabolically labeled with [¹⁴C]GlcNAc or [³H]inositol ([³H]Ins) under permissive (a, Gal or b, 30 °C) and nonpermissive (a, Glc or b, 37 °C) conditions. Extracted lipids were TLC separated and detected by fluorography. (c,d) [¹⁴C]GlcNAc-labeled lipids isolated from gpi18 and mcd4 cells were subjected to chemical and enzymatic treatments prior to TLC separation. In all panels, the positions of known Man₁(Etn-P)-GPI and Man₂-GPI species that accumulate in gpi18 and mcd4 are shown. These analyses confirm the presence of a Man₁-GPI (black star) accumulating in both gpi18 and mcd4 cells. TLC separation for a, b, c, and d was performed in solvents B, C, B, and D, respectively (see Isolation and separation of GPI lipid intermediates). PLC, phospholipase C; base, mild base hydrolysis; αM, jack bean α-mannosidase; M, mannose; E, ethanolamine phosphate; G, glucosamine; PI, phosphatidylinositol.

Fig 6

Accumulation of un-substituted GPI intermediates in later-stage GPI synthesis mutants. (a) TLC separation (in solvent E) of [¹⁴C]GlcNAc-labeled GPI intermediates that accumulate in gpi18, gpi10, and smp3 mutants under nonpermissive conditions. The positions of putative un-substituted Man₁-, Man₂-, and Man₃-GPIs are denoted with a black star, diamond and circle, respectively. The structures and positions of known GPI intermediates are also shown. (b,c) TLC separation (in solvent B) of [¹⁴C]GlcNAc-labeled lipids from gpi10 and smp3 mutants after chemical or enzymatic treatment. (d) Characterization of a putative un-substituted Man₃-GPI (black circle) that accumulates in smp3 cells by digestion with α1,6 and α1,2 mannosidases. TLC separation was performed with solvent B. PLC, phospholipase C; base, mild base hydrolysis; αM, jack bean α-mannosidase, M, mannose; E, ethanolamine phosphate; G, glucosamine; PI, phosphatidylinositol.

Fig 7

A revised model for yeast GPI glycan synthesis. A branched GPI glycan synthesis pathway is suggested from the structures of lipids that accumulate in various conditional yeast GPI synthesis mutants. Two new lipids lacking Etn-P were identified in this study, a Man₁-GPI (blue highlight) and a Man₃-GPI (pink highlight). Formation of these lipids suggests that Mcd4p and Gpi18p do not act in a specific order and provides a route to the putative synthesis of Man₄-GPIs that lack Etn-P side-branches. It is currently unclear whether structures lacking Etn-P on Man-1 are ultimately converted to the complete precursor CP2. Arrow thickness reflects relative differences in lipid abundance observed in each pathway branch.