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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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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.
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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.

Mentions: Deletion of TED1 causes ER retention of Gas1 (Haass et al., 2007) and therewith induces a UPR (Jonikas et al., 2009). We therefore decided to verify whether cdc1 cells show similar ER retention of GPI proteins and display signs of ER stress. Gas1 is an abundant GPI-anchored β1,3-transglucosidase, which, after extensive N- and O-glycosylation in the ER, runs with an apparent molecular weight (MW) of 105 kDa on SDS–PAGE, but at 125 kDa after elongation of its glycans in the Golgi (Fankhauser and Conzelmann, 1991). Pulse-chase experiments shown in Figure 5A indicate that cdc1 cells export Gas1 out of the ER with normal kinetics, whereas its export is delayed in ted1∆ mutants. Similarly, as shown by Figure 5B, Western blotting of extracts of cdc1 cells having been at 24, 30, 33, or 37°C during 4 h show no significant accumulation of an immature ER form of Gas1, much in contrast to bst1∆ and per1∆ cells (Figure 1). Ccw12 is a GPI-CWP undergoing extensive elongation of its three N-glycans in the Golgi, a process that raises the apparent MW of its 50- and 58-kDa forms to >200 kDa (Ragni et al., 2007). The disappearance of the 50/58-kDa forms of Ccw12 upon addition of cycloheximide was complete within 15 min and followed similar kinetics in cdc1 and WT cells, indicating that ER-to-Golgi transport and elongation of glycans are not compromised in the cdc1 mutant (Figure S2).


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: Deletion of TED1 causes ER retention of Gas1 (Haass et al., 2007) and therewith induces a UPR (Jonikas et al., 2009). We therefore decided to verify whether cdc1 cells show similar ER retention of GPI proteins and display signs of ER stress. Gas1 is an abundant GPI-anchored β1,3-transglucosidase, which, after extensive N- and O-glycosylation in the ER, runs with an apparent molecular weight (MW) of 105 kDa on SDS–PAGE, but at 125 kDa after elongation of its glycans in the Golgi (Fankhauser and Conzelmann, 1991). Pulse-chase experiments shown in Figure 5A indicate that cdc1 cells export Gas1 out of the ER with normal kinetics, whereas its export is delayed in ted1∆ mutants. Similarly, as shown by Figure 5B, Western blotting of extracts of cdc1 cells having been at 24, 30, 33, or 37°C during 4 h show no significant accumulation of an immature ER form of Gas1, much in contrast to bst1∆ and per1∆ cells (Figure 1). Ccw12 is a GPI-CWP undergoing extensive elongation of its three N-glycans in the Golgi, a process that raises the apparent MW of its 50- and 58-kDa forms to >200 kDa (Ragni et al., 2007). The disappearance of the 50/58-kDa forms of Ccw12 upon addition of cycloheximide was complete within 15 min and followed similar kinetics in cdc1 and WT cells, indicating that ER-to-Golgi transport and elongation of glycans are not compromised in the cdc1 mutant (Figure S2).

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
Related in: MedlinePlus