Roy A, Lu C F, Marykwas D L, Lipke P N, Kurjan J
Department of Biological Sciences, Columbia University, New York, New York 10027.
Mol Cell Biol. 1991 Aug;11(8):4196-206. doi: 10.1128/mcb.11.8.4196-4206.1991.
Saccharomyces cerevisiae a and alpha cells express the complementary cell surface glycoproteins a-agglutinin and alpha-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating. Treatment of S. cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available. To characterise a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAI). Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called aga1. The aga1 mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants. The AGA1 gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors. Active a-agglutinin binding subunit is secreted by aga1 mutants, indicating that AGA1 is involved in cells surface attachment of a-agglutinin. This result suggests that AGA1 encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts. Unexpectedly, the AGA1 transcript was expressed and induced by pheromone in both a and alpha cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.
酿酒酵母的a型细胞和α型细胞分别表达互补的细胞表面糖蛋白a-凝集素和α-凝集素，它们相互作用以促进交配过程中的细胞聚集。用还原剂处理酿酒酵母a型细胞会释放a-凝集素的结合亚基，该亚基已被纯化和表征；关于a-凝集素整体结构的生化信息很少。为了表征a-凝集素的结构和功能，我们采用遗传方法克隆了一个a-凝集素结构基因（AGAI）。分离出具有a特异性凝集缺陷的突变体，其中大多数属于一个单一的互补群，称为aga1。aga1突变体表现出野生型信息素的产生和反应，在固体培养基上有效交配，而在液体培养基上存在交配缺陷；这些表型是凝集素突变体的特征。通过互补作用克隆了AGA1基因；基因序列表明它可以编码一个含有725个氨基酸的蛋白质，丝氨酸和苏氨酸含量高，有一个推定的N端信号序列，以及一个C端疏水序列，类似于与糖基磷脂酰肌醇锚定连接的信号。活性a-凝集素结合亚基由aga1突变体分泌，表明AGA1参与a-凝集素在细胞表面的附着。这一结果表明AGA1编码的蛋白质与来自其他出芽酵母的a-凝集素类似物的核心亚基具有功能相似性。出乎意料的是，AGA1转录本在a型细胞和α型细胞中均由信息素表达和诱导，这表明活性a-凝集素的a特异性表达仅源于a-凝集素结合亚基的a特异性调控。
