Vazquez Hector M, Vionnet Christine, Roubaty Carole, Conzelmann Andreas
Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland.
Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland
Mol Biol Cell. 2014 Nov 1;25(21):3375-88. doi: 10.1091/mbc.E14-06-1033. Epub 2014 Aug 27.
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.
据报道，温度敏感型cdc1（ts）突变体在G2早期转移到30°C时会停止细胞周期，也就是说，作为已完成DNA复制但无法复制纺锤极体的小芽细胞。最近的一份报告显示，CDC1的人类同源物PGAP5作为一种磷酸二酯酶，从糖基磷脂酰肌醇（GPI）锚定物的甘露糖2上去除磷酸乙醇胺（EtN-P），从而允许GPI蛋白从内质网（ER）到高尔基体的有效运输。我们发现，在mcd4∆细胞中可以缺失必需的CDC1基因，mcd4∆细胞不会将EtN-P连接到GPI锚定物的甘露糖1上，这表明Cdc1去除了Mcd4添加的EtN-P。Cdc1-314（ts）突变体不会在ER中积累GPI蛋白，但在分泌途径后期有部分分泌阻滞。生长测试和cdc1-314（ts）的遗传相互作用图谱确定了一个明显的细胞壁缺陷。渗透支持可恢复GPI蛋白分泌和肌动蛋白极化，但不能恢复生长。cdc1-314（ts）突变体的细胞壁含有大量GPI蛋白，这些蛋白很容易被β-葡聚糖酶释放，且不与细胞壁β1,6-葡聚糖连接，并保留其原始的GPI锚定脂质。这表明cdc1-314（ts）假定的转糖基酶Dfg5和Dcw1将GPI蛋白低效地转移到细胞壁β1,6-葡聚糖上。
