Molecular analysis of Chs3p participation in chitin synthase III activity
Molecular analysis of Chs3p participation in chitin synthase III activity
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DOI：
10.1046/j.1432-1327.1998.2560419.x
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发表时间：
1998-09-01
期刊：
影响因子：
--
通讯作者：
Roncero, C
中科院分区：
文献类型：
--
作者：
Cos, T;Ford, RA;Roncero, C
几丁质是酿酒酵母细胞壁的次要但必需的组分，在营养生命周期中的隔膜形成中以及在有性周期中的接合和孢子细胞壁合成中具有功能。在酵母中存在的三种几丁质酶中，几丁质合成酶III（CSIII）负责合成细胞中发现的大部分几丁质，包括早期出芽时的几丁质环、散布在细胞壁中的几丁质和在性周期期间沉积的几丁质。我们已经标记了Chs3p，CSIII的推定催化亚基，流感病毒血凝素的免疫反应性表位，以遵循蛋白质的表达。Chs3p的转录和翻译水平与体内功能之间几乎没有相关性，这支持了我们先前的结论，即CSIII的调控发生在翻译后水平。为了鉴定参与催化或调节的蛋白质的可能区域，在几丁质酶的QRRRW“特征序列”中产生突变。精氨酸残基突变的Chs3p，并在Chs1p和Chs2p，导致在体内的功能和酶活性的损失。在Chs3p中与谷氨酰胺相邻的丝氨酸残基中的突变导致CSIII活性中度降低的体内功能丧失，表明丝氨酸残基在几丁质生物合成中的调节作用。在独特的亲水性羧基末端区域的Chs3p的几个截短确定了一个约25个氨基酸的序列，这是所需的功能和体外活性。由于该区域不存在于Chs1或Chs2中，因此它可能参与CSIII的特异性调节。
Chitin is a minor but essential component of the cell wall of Saccharomyces cerevisiae, with functions in septum formation in the vegetative life cycle and also in conjugation and spore cell-wall synthesis in the sexual cycle. Of the three chitin synthases present in yeast, chitin synthase III (CSIII) is responsible for the synthesis of most of the chitin found in the cell, including a chitin ring at early budding, chitin interspersed in the cell wall, and chitin laid down during the sexual cycle. We have tagged Chs3p, the putative catalytic subunit of CSIII, with the immunoreactive epitope of influenza virus hemagglutinin to follow expression of the protein. Little correlation was found between the levels of transcription and translation of Chs3p and in vivo function, supporting our previous conclusion that regulation of CSIII occurs at the posttranslational level. To identify possible regions of the protein involved in catalysis or regulation, mutations were generated in the QRRRW 'signature sequence' of chitin synthases. Arginine residue mutations in Chs3p, and in Chs1p and Chs2p, resulted in a loss of both function in vivo and enzymatic activity. Mutations in a serine residue adjacent to glutamine in Chs3p caused loss of function in vivo with a moderate decrease in CSIII activity, suggesting a regulatory role for the serine residue in chitin biosynthesis. Several truncations in the unique hydrophilic carboxy-terminal region of Chs3p identified a sequence of about 25 amino acids that is required for both function and in vitro activity. Since this region is not present in Chs1 or Chs2,, it may be involved in the specific regulation of CSIII.