Longtine M S, Fares H, Pringle J R
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Cell Biol. 1998 Nov 2;143(3):719-36. doi: 10.1083/jcb.143.3.719.
To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother-bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Delta vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Delta cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616).
为了鉴定酿酒酵母中与septin相互作用的蛋白，我们筛选了与cdc12 septin突变具有合成致死性的突变。鉴定出的其中一个基因是GIN4，它编码一种蛋白激酶，与酿酒酵母中的Hsl1p/Nik1p和Ycl024Wp以及粟酒裂殖酵母中的Nim1p/Cdr1p和Cdr2p相关。Gin4p激酶结构域与Cdc3p septin的COOH末端部分显示出双杂交相互作用，并且Gin4p与septin在母-芽颈处共定位。这种定位依赖于septin和Gin4p的COOH末端（非激酶）区域，并且该COOH末端区域的过量表达导致septin组织的丧失和相关的形态发生缺陷。单独缺失YCL024W或与缺失GIN4联合缺失，我们均未检测到影响。缺失GIN4并不致死，但导致septin的显著重组，伴有形态发生异常和细胞分离缺陷；然而，值得注意的是，胞质分裂似乎有效地发生了。另外两种以septin依赖方式定位于颈部的蛋白也显示出类似的重组，并且似乎在很大程度上仍保持功能。在gin4Δ营养细胞中观察到的septin组织类似于在响应交配信息素的细胞中正常看到的情况，并且在这些细胞中未检测到与septin相关的Gin4p。在gin4Δ细胞和响应信息素的细胞中观察到的septin组织似乎支持Field等人（Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616）先前提出的septin组织模型的某些方面。
