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

Abstract

Temperature-sensitive cdc1(ts) mutants are reported to stop the cell cycle upon a shift to 30°C in early G2, that is, as small budded cells having completed DNA replication but unable to duplicate the spindle pole body. A recent report showed that PGAP5, a human homologue of CDC1, acts as a phosphodiesterase removing an ethanolamine phosphate (EtN-P) from mannose 2 of the glycosylphosphatidylinositol (GPI) anchor, thus permitting efficient endoplasmic reticulum (ER)-to-Golgi transport of GPI proteins. We find that the essential CDC1 gene can be deleted in mcd4∆ cells, which do not attach EtN-P to mannose 1 of the GPI anchor, suggesting that Cdc1 removes the EtN-P added by Mcd4. Cdc1-314(ts) mutants do not accumulate GPI proteins in the ER but have a partial secretion block later in the secretory pathway. Growth tests and the genetic interaction profile of cdc1-314(ts) pinpoint a distinct cell wall defect. Osmotic support restores GPI protein secretion and actin polarization but not growth. Cell walls of cdc1-314(ts) mutants contain large amounts of GPI proteins that are easily released by β-glucanases and not attached to cell wall β1,6-glucans and that retain their original GPI anchor lipid. This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314(ts) transfer GPI proteins to cell wall β1,6-glucans inefficiently.

FIGURE 1:

GPI anchor remodeling and subsequent attachment to the cell wall. Essential genes are indicated in blue. Mature GPI anchor lipids (left) contain EtN-Ps on Man1, Man2, and Man3; their addition is catalyzed by MCD4, GPI7, and GPI13, respectively. After the protein is attached to the GPI lipid by the transamidase complex, the lipid moiety is remodeled by Bst1, Per1, Gup1, and Cwh43 (middle). At some time during lipid remodeling, EtN-Ps may be removed from Man1 and Man2. At the plasma membrane, Man1 of the anchor of GPI-CWPs is transferred and covalently linked to β1,6-glucans. This reaction is presumably catalyzed by Dcw1 and Dfg5.

FIGURE 2:

The essential CDC1 gene can be deleted in the mcd4∆ mutant, which fails to add EtN-P onto Man1 of the GPI anchor. A diploid mcd4∆/MCD4 cdc1∆/CDC1 strain harboring vectors expressing GPI10 from Trypanosoma brucei (TbGPI10) (LEU2) and yeast CDC1 (URA3) was sporulated and dissected. A tetratype tetrad is shown at the top left; haploid offspring carrying mcd4∆ are producing very small colonies. The four colonies of this tetrad were grown, and 10-fold dilutions of cells were spotted on SC media with indicated supplements and grown at 30°C for 2–3 d.

FIGURE 3:

Genetic interactions of cdc1. (A) Fourfold dilutions of cdc1, cdc1 harboring WT CDC1, or the functionally dead cdc1^D144A allele were incubated at 24 or 30°C. (B) Small areas from plates used to obtain colony sizes and Z-scores for cdc1/geneX∆ double mutants are shown. The upper three and lower three areas each show the same mutants selected either at 24, 26, or 30°C. Two double mutants (in quadruplicate) showing significant negative interaction at 24 as well as 26°C, or only at 26°C, are boxed in yellow and red, respectively, one showing positive genetic interaction is boxed in blue. The same mutants at the temperatures at which they do not get significant Z-scores are in dotted-line boxes. (C) Z-scores observed at 24 and 26°C of ∼5500 deletion strains remaining after the first filtering (see Materials and Methods) are plotted. Significant hits (Z-scores > 2.7; p values < 0.01) found only at 24°C are shown in green, hits seen only at 26°C in red, and hits found in both screens in yellow. (D–F) Negative interactions found at 24 and 26°C (D and E) and positive ones found at 30°C (F) were manually clustered into functional categories. Functional classes enriched at both 24 and 26°C are in bold. Only interactions remaining after a second filtering (see Materials and Methods) are reported.

FIGURE 4:

Stability of Cdc1-314 and Cdc1 proteins. (A) WT cells harboring HA-tagged Cdc1 or Cdc1-314 proteins were incubated at 24°C overnight (ON) and then shifted to 30 and 37°C for the indicated times. Thereafter cell extracts were subjected to SDS–PAGE and Western blotting using anti-HA antibodies. Adh1 was detected as a loading control. Signals of two biological replicates were averaged and normalized using the Adh1 signals and reference samples (see Materials and Methods). (B) Stability of HA-tagged Cdc1 and Cdc1-314 proteins in cells having gene deletions that were identified as suppressors. Cells were incubated overnight at 30 and 37°C before being processed as in A. Quantifications of A and B are not directly comparable, as normalizations were done only within the experiments of each panel.

FIGURE 5:

Remodeling and export of GPI proteins and induction of UPR in cdc1. (A) WT and mutants were pulse labeled with [³⁵S]Met/Cys and chased for indicated times. Gas1 was immunoprecipitated and detected by autoradiography. (B) Western blots using anti-Gas1 antibody of total protein extracts from strains grown for 4 h at 24, 30, 33, or 37°C. (C) WT and cdc1 carrying an UPRE-lacZ plasmid were preincubated for 5 or 3 h at 27 or 30°C, then further incubated in the presence or absence of DTT for 2 h. Thereafter UPR induction was assessed by measuring β-galactosidase activity, which was plotted as absolute activity (upper plots) or fold induction caused by DTT (lower plots). (D) WT or cdc1 cells were preincubated at 37°C for 60 min (upper panel) or 10 min (lower panel) and then labeled with [³H]myo-inositol for 2 h. Anchor peptides from the total of SDS-extractable GPI proteins were isolated, lipid moieties were released by HNO₂ treatment, subjected or not to mild alkaline deacylation with NaOH, resolved by TLC, and detected by autoradiography. No lipids were seen when HNO₂ treatment was omitted, indicating that only GPI anchor lipids had been isolated. An aliquot of free lipids (FL), not attached to GPI anchors, was run on the same TLC for comparison. (E) Lipid extracts from cells radiolabeled for the upper panel in D were analyzed by TLC and autoradiography before and after deacylation with NaOH. (F) Lipid extracts from [¹⁴C]serine-labeled cells preincubated and labeled at 30°C for 5 h were analyzed as in E.

FIGURE 6:

cdc1 cells have fragile cell walls. (A) Fourfold serial dilutions of the indicated strains were spotted onto YPD containing low concentrations of cell wall–perturbing agents and incubated at 30°C (mcd4∆ strains) or 27°C (all others). (B) Same as A but plates contained sorbitol (1 or 1.4 M) and were incubated at indicated temperatures. The same picture as in A was also found when the identical assays were repeated on SC medium (unpublished data). (C) WT and cdc1 were also tested for resistance to cell wall–perturbing agents in the presence of 1.4 M sorbitol, at 30°C for the plate containing CFW and at 32°C for all others. (D) CFW staining of WT and cdc1 cells grown in YPD at 30°C for 16 h. The pictures are directly comparable, as they were taken using the same exposure time and were processed in the same way. All plates were incubated 2 d except for the ones with mcd4∆ strains (4 d).

FIGURE 7:

Sorbitol normalizes surface transport of GPI proteins and actin depolarization in cdc1 cells. WT and cdc1 cells expressing Gas1-GFP (A) or Crh2-GFP (B) under their own promoters and present on centromeric plasmids were grown overnight at the indicated temperatures without or with 1 M sorbitol (+ 1MS). Cells were viewed under a fluorescence microscope. Magnification is the same in all pictures; scale bars: 5 μm. (C) actin patches and cables were stained with rhodamine phalloidin in cells grown overnight under the indicated conditions. (D and E) Actin polarization in cells grown overnight at 33 (D) or 37°C (E) was quantified by measuring the phalloidin fluorescence density in buds and their mothers and then calculating the bud/mother density ratios. Each dot represents a budded cell; bud/mother density ratios are plotted as a function of the bud/mother size ratio and data were subjected to linear regression analysis. Note that these fluorescence density ratios are quite small, because they do not account for the volume but only the area occupied by buds and mothers.

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.