Nakase Y, Nakamura T, Hirata A, Routt S M, Skinner H B, Bankaitis V A, Shimoda C
Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan.
Mol Biol Cell. 2001 Apr;12(4):901-17. doi: 10.1091/mbc.12.4.901.
The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.
粟酒裂殖酵母spo20-KC104突变最初是在对孢子形成缺陷型突变体的筛选中分离得到的，spo20-KC104突变体表现出温度敏感型生长。在此，我们报告粟酒裂殖酵母spo20(+)对于裂殖酵母细胞的生存能力至关重要，并且在整个生命周期中持续表达。我们还证明spo20(+)基因产物在结构上与酿酒酵母的Sec14同源，Sec14是芽殖酵母的主要磷脂酰肌醇转移蛋白。这种结构同源性转化为显著程度的功能相关性，因为相互互补实验表明每种蛋白质都能够履行另一种蛋白质的基本功能。此外，生化实验表明，与Sec14一样，Spo20是一种磷脂酰肌醇/磷脂酰胆碱转移蛋白。我们证明spo20-KC104突变体在限制温度下积累异常的高尔基体潴泡，这表明Spo20是营养细胞中高尔基体分泌功能所必需的。然而，在缺乏Spo20的裂殖酵母中观察到的第二种表型是细胞分裂在细胞分离完成之前停滞。与Spo20在控制营养生长细胞中的细胞隔膜形成中起直接作用一致，定位实验表明Spo20优先定位于裂殖酵母细胞的细胞极和隔膜部位。我们还报告说，当裂殖酵母受到氮饥饿挑战时，Spo20会转移到细胞核。这种核定位在接合和减数分裂过程中持续存在。减数分裂完成后，Spo20转移到前孢子膜，并且在spo20-KC104细胞中前孢子膜的组装是异常的。在这种突变体中，相当一部分正在形成的前孢子未能包裹单倍体细胞核。我们的结果表明，Spo20除了在调节高尔基体分泌功能方面的公认作用外，还调节特殊膜结构的形成。
