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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.

Vazquez HM, Vionnet C, Roubaty C, Conzelmann A - Mol. Biol. Cell (2014)

Bottom Line: 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.This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314(ts) transfer GPI proteins to cell wall β1,6-glucans inefficiently.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland.

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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.
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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.

Mentions: Because ERAD mutants rescued growth of cdc1 (Figure 3F), we hypothesized that ERAD mutants may stabilize Cdc1-314. To verify this, we expressed HA-Cdc1 or HA-Cdc1-314, that is, tagged versions carrying the hemagglutinin-derived HA epitope (HA) under the native promoter from a centromeric vector in WT cells and quantified the amounts of HA-tagged protein after shifting cells from 24° to 30° or 37°C, as shown in Figure 4A. In cells growing at 24°C, Cdc1-314 already was 3.8-fold less abundant than WT Cdc1. A shift to 30°C did not destabilize Cdc1-314, although cells depending only on mutant protein do not grow at this temperature (Figure 3A). When cells were shifted to 37°C, a third of Cdc1-314 was degraded within 15–30 min, but the protein remained stable thereafter. The same temperature shifts reduced the amounts of WT Cdc1, but more slowly, so that it took more than 4 h to reach equilibrium levels.


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.

Vazquez HM, Vionnet C, Roubaty C, Conzelmann A - Mol. Biol. Cell (2014)

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.
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Related In: Results  -  Collection

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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.
Mentions: Because ERAD mutants rescued growth of cdc1 (Figure 3F), we hypothesized that ERAD mutants may stabilize Cdc1-314. To verify this, we expressed HA-Cdc1 or HA-Cdc1-314, that is, tagged versions carrying the hemagglutinin-derived HA epitope (HA) under the native promoter from a centromeric vector in WT cells and quantified the amounts of HA-tagged protein after shifting cells from 24° to 30° or 37°C, as shown in Figure 4A. In cells growing at 24°C, Cdc1-314 already was 3.8-fold less abundant than WT Cdc1. A shift to 30°C did not destabilize Cdc1-314, although cells depending only on mutant protein do not grow at this temperature (Figure 3A). When cells were shifted to 37°C, a third of Cdc1-314 was degraded within 15–30 min, but the protein remained stable thereafter. The same temperature shifts reduced the amounts of WT Cdc1, but more slowly, so that it took more than 4 h to reach equilibrium levels.

Bottom Line: 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.This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314(ts) transfer GPI proteins to cell wall β1,6-glucans inefficiently.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland.

Show MeSH