Cabib Enrico, Farkas Vladimir, Kosík Ondrej, Blanco Noelia, Arroyo Javier, McPhie Peter
Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.
J Biol Chem. 2008 Oct 31;283(44):29859-72. doi: 10.1074/jbc.M804274200. Epub 2008 Aug 11.
The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to beta(1-3)glucose branches of beta(1-6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to beta(1-6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established.
对于每一个新形成的连接，预先存在的连接要被破坏，以适应质膜外不存在自由能量来源的情况。先前的研究表明，Crh1p和Crh2p这两种假定的转糖基酶，对于出芽酵母细胞壁中几丁质与β(1-6)葡聚糖的β(1-3)葡萄糖分支的连接是必需的。为了在体内和体外探索连接反应，我们使用荧光磺基罗丹明连接的海带寡糖作为人工几丁质受体。在体内，在芽痕处检测到荧光，在细胞轮廓处荧光水平较低，两者都依赖于CRH基因。在洋地黄皂苷通透的细胞中也显示出连接反应，以UDP-N-乙酰葡糖胺作为新生几丁质产生的底物。这里核苷酸和Crh蛋白都是必需的。一个过表达Crh1p的gas1突变体在完整细胞和通透细胞中都显示出非常高的荧光。在后者中，在没有UDP-GlcNAc的情况下，荧光仍以斑块形式掺入。当用磺基罗丹明寡糖孵育该菌株的分离细胞壁时，也显示出依赖Crhp的斑块状荧光，这些斑块被鉴定为芽痕。在所有这三个系统中，通过几丁质酶消化验证了荧光物质与几丁质的结合。此外，几丁质寡糖抑制细胞壁反应。这些结果表明，Crh蛋白通过将几丁质链转移到β(1-6)葡聚糖上起作用，在芽痕中观察到新的高活性。因此，转糖基化对细胞壁组装的重要性得到了有力证实。
