The role of glucosidase I (Cwh41p) in the biosynthesis of cell wall beta-1,6-glucan is indirect

Abstract

CWH41, a gene involved in the assembly of cell wall beta-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal alpha-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of beta-1,6-glucan, we constructed a double mutant, alg5Delta (lacking dolichol-P-glucose synthase) cwh41Delta, and found that it has the same phenotype as the alg5Delta single mutant. It contains wild-type levels of cell wall beta-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Delta single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the beta-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Delta). The triple mutant alg5Deltacwh41Deltakre6Delta is viable, whereas the double mutant cwh41Deltakre6Delta in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall beta-1,6-glucan, characteristic of the cwh41Deltakre1Delta double mutant, are not observed in the triple mutant alg5Deltacwh41Deltakre1Delta. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Delta strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall beta-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.

Figure 1

Calcofluor sensitivity test. Yeast cells were resuspended at 10⁶ cells/ml in sterile H₂O. Serial dilutions of 1-, 5-, 25-, and 125-fold were made in sterile water, and 4 μl of each dilution were spotted on YPD plates containing the indicated concentration of CFW. The plates were incubated for 3 d at 24°C before photography. Strains LCY21 (SEY6210, WT), LCY24 (cwh41Δ), LCY22 (alg5Δ), and LCY23 (alg5Δcwh41Δ) represent a tetrad from strain LCY 20.

Figure 2

N-glycosylation of Wbp1p. Total cell extracts from strains LCY21 (lane 1), LCY22 (lane2), LCY23 (lane 3), and LCY24 (lane 4) were analyzed by 8% SDS/PAGE and Western blot. Arrows indicate the number of glycosylation sites used. (A and B) Structure of the oligosaccharides, N-acetylglucosamine (closed squares), mannose (open circles), and glucose (closed inverted triangles).

Figure 3

Germination and growth of tetrads. Diploid strains were sporulated and dissected onto YPD plates and incubated at 24°C for the amount of time indicated. Tetrad types shown are parental ditype (PD), nonparental ditype (NPD), and tetratype (TT). The four spore progeny derived from each tetrad are indicated by the letters a–d to the left of each panel. (A) Diploid strain LCY45 (alg5Δ/alg5Δ, kre6Δ/KRE6, cwh41Δ/CWH41). (B) Diploid strain LCY80 (kre1Δ/KRE1, cwh41Δ/CWH41). (C) Diploid strain LCY65 (alg5Δ/alg5Δ, kre1Δ/KRE1, cwh41Δ/CWH41).

Figure 4

Levels of cell wall β-1,6-glucan. Alkali-insoluble β-1,6-glucan was extracted from cell wall preparations of various strains and quantified in micrograms per milligram of cell wall dry weight as described in MATERIALS AND METHODS. The data represent the mean ± SD of five independent experiments. Strains A: SEY6210 (wild type), TR92 (kre6Δ), LCY33(kre6Δalg5Δ), LCY47 (kre6Δcwh41Δalg5Δ); strains on B: HAB635(kre1Δ), LCY58 (kre1Δalg5Δ), LCY81 (kre1Δcwh41Δ), LCY66 (alg5Δkre1Δcwh41Δ).

Figure 5

Stability of Kre6p in the cwh41Δ mutant. Total cell extracts from strains LCY24 (cwh41Δ) and LCY23 (alg5Δcwh41Δ) were subject to 10% SDS-PAGE and Western blots, followed by quantification of the indicated proteins (see MATERIALS AND METHODS). Integration is expressed in arbitrary units. The same membrane was probed successively with antiserum against the hemagglutinin epitope tag of Kre6p (A) and Wbp1p (B).

Figure 6

Kre6p overexpression and CFW sensitivity of cwh41Δ mutant. The sensitivity test was done as described in the legend of Figure 1. Concentrations of CFW are indicated. Strains LCY24 (cwh41Δ) and LCY23 (alg5Δcwh41Δ) were transformed with either multicopy (YEp24) or centromeric (pRS315) plasmids containing KRE6 or KRE6-HA.