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