Cdc1 removes the ethanolamine phosphate of the first mannose of GPI anchors and thereby facilitates the integration of GPI proteins into the yeast cell wall.
Cdc1-314(ts) mutants do not accumulate GPI proteins in the ER but have a partial secretion block later in the secretory pathway.Osmotic support restores GPI protein secretion and actin polarization but not growth.This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314(ts) transfer GPI proteins to cell wall β1,6-glucans inefficiently.
Affiliation: Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland.
- Cell Cycle Proteins/genetics/metabolism*
- Cell Wall/genetics/metabolism*
- Saccharomyces cerevisiae Proteins/genetics/metabolism*
- Endoplasmic Reticulum/metabolism
- Membrane Glycoproteins/genetics/metabolism
- Membrane Proteins/genetics/metabolism
- Protein Transport/drug effects
- Saccharomyces cerevisiae/drug effects/genetics/metabolism
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Figure 8: The transfer of GPI-CWPs onto β1,6-glucans is compromised in cdc1 cells. (A) SDS-treated cell walls (10 OD600 equivalents) from the indicated strains harboring a plasmid with HA-Cwp2 and having been grown in presence or absence of 1 M sorbitol were digested with the specified glycosidases or control incubated, boiled in sample buffer, and centrifuged. Supernatants were loaded and separated in a 4–20% gradient SDS–PAGE. Cwp1 (top) and HA-Cwp2 (bottom) were detected on Western blots. SDS extracts from the same cells, as well as proteins secreted into the media corresponding to 0.5 and 10 OD600 equivalents, respectively, are shown in lanes 1–4 and 33–36. The specificity of the polyclonal anti-Cwp1 antibody is shown in lanes 5, 15, 27, and 32. The antibodies used do not react with the glycosidases used (lanes 14, 20, and 26). (B) Cells of indicated genotypes were metabolically labeled with [3H]myo-inositol, mechanically disrupted, and divided into two equal parts. One part was subjected to extensive delipidation using organic solvents, proteins were extracted by repeated boiling in SDS, and nonsoluble material was removed by centrifugation (SDS extractable); in the other part, cell walls were prepared by repeated SDS extraction and were further delipidated with organic solvent (cell wall associated). In both parts, the remaining, GPI protein–associated radioactivity was detected by scintillation counting. (C) Same as in B but cells were grown overnight and labeled in media containing 1 M sorbitol. All strains in B and C, except for cwp1∆, had the Y8205 genetic background. (D) Same as B. Average and SD of three independent experiments and nine measurements are shown for B, and two independent experiments and six measurements are shown for C and D.
In our experiments done with cells growing in SC medium at low pH (4.2), Cwp1 of WT cells was also released only by combined treatment of β1,3- and β1,6-glucanases and chitinase (Figure 8A, top panel). In contrast, Cwp1 of cdc1 cells was already released by incubation at 60°C alone, and its release was increased by single digestions with either β1,6- or β1,3-glucanase or chitinase. That higher amounts of Cwp1 are released from cdc1 cell walls than from WT cell walls is due to the well-known induction of CWP1 upon cell wall stress (Ram et al., 1998; García et al., 2004; Figure S5). This is apparent also in SDS extracts of cells (Figure 8A, lanes 1–4, top panel).