Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization
Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization
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作者：
Carotti, C;Ferrario, L;Popolo, L
几丁质合酶 III 对于缺乏 Gas1p（一种 β(1,3)-葡聚糖基转移酶）的细胞中几丁质水平的增加和细胞完整性至关重要。为了发现几丁质合成的上调是通过 Chs3p 的经典运输和激活途径还是通过替代途径进行，在这里我们研究了 GAS1 和 CHS4-5-6-7 基因失活的影响。所有双无效突变体都显示出温度敏感的细胞裂解表型，这种表型可以通过渗透稳定剂的存在来抑制。在30℃的液体YEPD中，chs4Deltagas1Delta、chs5Deltagas1Delta、chs6Deltagas1Delta和chs7Deltagas1Delta突变体无法生长，而它们在基本培养基中生长非常缓慢并且表现出低活力。高渗透压抑制了缺陷表型并恢复了生长。在chs4gas1、chs5gas1和chs7gas1中，几丁质水平没有增加，仅降低到10%，而在chs6gas1中，gas1的值降低到20-40%。为了研究几丁质合成的上调可能发生在什么水平，监测了 mRNA 水平。 CHS4-5-6-7的表达在gas1A中没有显着变化。在表达 HA 标记形式的菌株中，检查了 Chs3p 和 Chs5p 的定位。在gas1突变体中，荧光模式受到影响，芽中的蛋白质出现异常。结果表明：（a）CHS4-7基因的功能是gas1突变体中几丁质超积累和细胞完整性所必需的； (b) 同源基因不取代其功能； (c)CHS4-7基因的调节不发生在转录水平。控制几丁质合成的位置对于保护芽免于裂解可能很重要。版权所有 (C) 2002 John Wiley Sons, Ltd.
Chitin synthase III is essential for the increase in chitin level and for cell integrity in cells lacking Gas1p, a beta(1,3)-glucanosyltransferase. In order to discover whether the upregulation of chitin synthesis proceeds through the canonical transport and activation pathway of Chs3p or through an alternative one, here we studied the effects of the inactivation of the GAS1 and CHS4-5-6-7 genes. All the double-null mutants showed a temperature-sensitive cell lysis phenotype that could be suppressed by the presence of an osmotic stabilizer. In liquid YEPD at 30degreesC, chs4Delta gas1Delta, chs5Delta gas1Delta, chs6Deltagas1Delta and chs7Deltagas1Delta mutants were unable to grow, whereas they grew very slowly in minimal medium and showed low viability. High osmolarity suppressed the defective phenotype and restored growth. In chs4 gas1, chs5 gas1 and chs7 gas1, chitin levels did not increase and were reduced to only 10%, while in chs6 gas1 the value of gas1 was reduced to 20-40%. To investigate at which level the upregulation of chitin synthesis could occur, mRNA levels were monitored. The expression of CHS4-5-6-7 did not change significantly in gas1A. In strains expressing HA-tagged forms, the localization of Chs3p and Chs5p was examined. In the gas1 mutant the fluorescence pattern was affected and the proteins appeared abnormally present in the bud. The results indicate that: (a) the function of the CHS4-7 genes is required for chitin hyperaccumulation in gas1 mutant and for cell integrity; (b) homologous genes do not replace their function; (c) the regulation of CHS4-7 genes does not occur at transcriptional level. Control of the position of chitin synthesis could be important in protecting the bud from lysis. Copyright (C) 2002 John Wiley Sons, Ltd.