Elia L, Marsh L
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Cell Biol. 1996 Nov;135(3):741-51. doi: 10.1083/jcb.135.3.741.
Though early stages of yeast conjugation are well-mimicked by treatment with pheromones, the final degradation of the cell wall and membrane fusion of mating that leads to cytoplasmic mixing may require separate signals. Mutations that blocked cell fusion during mating in Saccharomyces cerevisiae were identified in a multipartite screen. The three tightest mutations proved to be partial-function alleles of the ABC-transporter gene STE6 required for transport of a-factor. The ste6(cefl-1) allele was recovered and sequenced. The ste6(cefl-1) allele contained a stop codon predicted to truncate Ste6 at amino acid residue 862 (of 1290). The ste6(cef) mutations reduced, but did not eliminate, expression of a-factor. Light and electron microscopy revealed that unlike ste6 null mutations which block mating before the formation of mating pairs, the ste6(cef) (cell fusion) alleles permitted early steps in mating to proceed normally but blocked at a late stage in conjugation where mating partners were encased by a single cell wall and separated by only a thin layer of cell wall material we term the fusion wall. Morphologically the prezygotes appeared symmetrical with successful cell wall fusion at the periphery of the region of cell contact. Responses to a-factor were efficiently induced in partner cells under mating conditions as expected given the symmetric appearance of the prezygotes. A strain expressing a ste6(K1093A) mutation that conferred export of a twofold to fourfold higher level of a-factor than ste6(cef) did not accumulate prezygotes during mating which could indicate a tight threshold of a-factor signaling required for mating. However, mating to an sst2 partner which has a greatly increased sensitivity to a-factor did not suppress the fusion defect of a ste6(cef) strain. Overexpression of the structural gene for a-factor also did not suppress the fusion defect. It is possible that a-factor or STE6 play more complex roles in cell fusion.
尽管用信息素处理能很好地模拟酵母接合的早期阶段，但导致细胞质混合的细胞壁最终降解和交配时的膜融合可能需要单独的信号。在一项多部分筛选中，鉴定出了酿酒酵母交配过程中阻止细胞融合的突变。最紧密的三个突变被证明是a因子运输所需的ABC转运蛋白基因STE6的部分功能等位基因。回收了ste6(cefl-1)等位基因并进行了测序。ste6(cefl-1)等位基因包含一个终止密码子，预计会在1290个氨基酸残基中的第862位截断Ste6。ste6(cef)突变降低但没有消除a因子的表达。光学显微镜和电子显微镜显示，与在交配对形成之前阻止交配的ste6无效突变不同，ste6(cef)（细胞融合）等位基因允许交配的早期步骤正常进行，但在接合的后期受阻，此时交配伙伴被单个细胞壁包裹，仅被一层我们称为融合壁的薄细胞壁物质分隔。形态上，合子前体在细胞接触区域的周边似乎对称，细胞壁成功融合。鉴于合子前体的对称外观，在交配条件下，伴侣细胞中对a因子的反应如预期那样被有效诱导。表达ste6(K1093A)突变的菌株，其a因子的输出水平比ste6(cef)高两到四倍，在交配过程中没有积累合子前体，这可能表明交配所需的a因子信号存在严格的阈值。然而，与对a因子敏感性大大增加的sst2伴侣交配并不能抑制ste6(cef)菌株的融合缺陷。a因子结构基因的过表达也不能抑制融合缺陷。a因子或STE6在细胞融合中可能发挥更复杂的作用。
