Mitchell D A, Sprague G F
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
Mol Cell Biol. 2001 Jan;21(2):488-500. doi: 10.1128/MCB.21.2.488-500.2001.
The Saccharomyces cerevisiae p21-activated kinases, Ste20p and Cla4p, have individual functions but appear to share an essential function(s) as well because a strain lacking both kinases is inviable. To learn more about the shared function, we sought new mutations that were lethal in the absence of CLA4. This approach led to the identification of at least 10 complementation groups designated NCS (need CLA4 to survive). As for ste20 cla4-75 mutants, most ncs cla4-75 double mutants were defective for septin localization during budding. One group, NCS1/RRD1 (YIL153w), did not confer this defect, however, and we investigated its function further. ncs1Delta cla4Delta cells arrested with elongated buds and short mitotic spindles. The morphological defects and lethality were suppressed by mutations that abrogate the cell cycle morphogenetic checkpoint, CDC28Y19F or swe1Delta. The connection to the cell cycle may be direct, as we detected a Cla4p-Cdc28p complex. NCS1 encodes a protein with significant similarity to a mammalian phosphotyrosyl phosphatase activator (PTPA) regulatory subunit for type 2A protein phosphatases (PP2As). Genetic and biochemical evidence suggested that the phosphatase Sit4p is a target for Ncs1p. First, CLA4 and SIT4 were synthetically lethal. Second, Ncs1p and its yeast paralog, Noh1p (Rrd2p), bound to the catalytic domain of Sit4p in vitro, and Ncs1p could be immunoprecipitated with Sit4p but not with another PP2A (Pph21p) from yeast cell extracts. Strains lacking both NCS1 and NOH1 were inviable and arrested as unbudded cells, implying that PTPA function is required for proper G(1) progression.
酿酒酵母的p21激活激酶Ste20p和Cla4p具有各自的功能，但似乎也共享一项基本功能，因为缺乏这两种激酶的菌株无法存活。为了更深入了解这种共享功能，我们寻找了在缺乏CLA4时具有致死性的新突变。这种方法导致鉴定出至少10个互补群，命名为NCS（需要CLA4才能存活）。至于ste20 cla4 - 75突变体，大多数ncs cla4 - 75双突变体在出芽过程中septin定位存在缺陷。然而，有一组NCS1/RRD1（YIL153w）并未导致这种缺陷，我们进一步研究了其功能。ncs1Δcla4Δ细胞因芽伸长和有丝分裂纺锤体短而停滞。废除细胞周期形态发生检查点的突变CDC28Y19F或swe1Δ可抑制形态缺陷和致死性。与细胞周期的联系可能是直接的，因为我们检测到Cla4p - Cdc28p复合物。NCS1编码一种与哺乳动物2A型蛋白磷酸酶（PP2A）的磷酸酪氨酸磷酸酶激活剂（PTPA）调节亚基具有显著相似性的蛋白质。遗传和生化证据表明磷酸酶Sit4p是Ncs1p的作用靶点。首先，CLA4和SIT4是合成致死的。其次，Ncs1p及其酵母旁系同源物Noh1p（Rrd2p）在体外与Sit4p的催化结构域结合，并且Ncs1p可以从酵母细胞提取物中与Sit4p一起免疫沉淀，但不能与另一种PP2A（Pph21p）一起免疫沉淀。同时缺乏NCS1和NOH1的菌株无法存活，并停滞为未出芽细胞，这意味着PTPA功能对于正常的G1期进程是必需的。
