Uridine diphosphate-glucose transport into the endoplasmic reticulum of Saccharomyces cerevisiae: in vivo and in vitro evidence

Abstract

It has been proposed that synthesis of beta-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose-dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP-glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER-containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP-glucose was temperature dependent and saturable with an apparent Km of 46 microM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP-N-acetylglucosamine did not enter these vesicles. Demonstration of UDP-glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP-glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Delta, lack cell wall beta-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Delta mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.

Figure 1

Rate of solutes accumulation within vesicles versus UDP–glucose concentration in the incubation medium. A P₂ vesicle fraction (see MATERIALS AND METHODS) (1.2 mg of protein) was incubated at 30°C for 3 min with 1 μCi of UDP–[³H]glucose plus different amounts of the unlabeled nucleotide sugar to give the desired final concentrations. Translocation results shown are the mean of two separate determinations. Inset, Double-reciprocal plot according to Lineweaver and Burk.

Figure 2

Cell-free assays of S. pombe GT expressed in S. cerevisiae. Microsomes from S. cerevisiae alg6 (PRY103) mutant cells transformed with p416GPD-gpt1⁺ (A) or p426GPD-gpt1⁺ (B) were incubated with UDP–[¹⁴C]glucose in the presence of 8 M urea-denatured thyroglobulin. Resulting Endo H-sensitive oligosaccharides were run on paper chromatography with solvent A. In panel C, thyroglobulin or detergent was omitted where indicated. Standards: 1, Glc₁Man₉GlcNAc; 2, Glc₁Man₈GlcNAc; and 3, Glc₁Man₇GlcNAc.

Figure 3

Intact-cell assay of S. pombe GT expressed in S. cerevisiae. S. cerevisiae alg6 mutant (PRY103) cells transformed with p426GPD-gpt1⁺ (A and B) or with p426GPD (C and D) were incubated with [¹⁴C]glucose for 15 min in the presence (A and C) or absence (B and D) of DNJ. Resulting Endo H-sensitive oligosaccharides were run on paper chromatography with solvent A. Standards: 1, Glc₁Man₉GlcNAc; 2, Glc₁Man₈GlcNAc; 3, Glc₁Man₇GlcNAc; 9, Man₉GlcNAc; 8, Man₈GlcNAc; and 7, Man₇GlcNAc.

Figure 4

Structural characterization of oligosaccharides. Compounds shown in Figure 3, A–C, were digested with α-mannosidase and run on paper chromatography with solvent B (A–C, respectively). Compound migrating as standard 3 in panel A was submitted to strong acid hydrolysis and run on paper chromatography with solvent C. Standards: 1, mannose; 2, ManGlcNAc; 3, Glc₁Man₄GlcNAc; and 4, glucose.

Figure 5

GT expression in GII minus cells. Alg5 (OCY2) or gls2-alg5 (OCY3) cells transformed with p425GPD-gpt1⁺ or p425 GPD were incubated for 15 min with [¹⁴C]glucose in the presence or absence of 5 mM DNJ. Endo H-liberated compounds were run on paper chromatography with solvent A. Material migrating between mid-Man₉GlcNAc and Man₈GlcNAc was submitted to strong acid hydrolysis and run on paper chromatography with solvent C. (A) alg5 cells with p425GPD-gpt 1⁺ incubated with DNJ and (B) gls2-alg5 with p425GPD-gpt⁺ cells incubated without DNJ. (C) alg5-gls2 cells with p425GPD incubated with DNJ. Standards: 1, mannose; and 2, glucose.

Figure 6

Effect of GT expression on the Kre5⁻ phenotype. Growth of HH3 cells (open circles), of a kre5::HIS3 mutant derived from it (OCY6) transformed with YEp352 (full circles), with pKRE5 (full squares) or with p425GPD-gpt1⁺ (open squares). Inset, RT-PCR analysis of RNA synthesized by two clones of kre5 mutant cells transformed with p425GPD-gpt1⁺. Primers used corresponded to the end fragments of the 665-bp 3′-terminus of gpt1⁺ (see MATERIALS AND METHODS). Lanes 1 and 2, complete system; lanes 3 and 4, omission of reverse transcriptase; lane 5, molecular weights markers.