The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle

Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.     

芽残酵母PHO85基因对细胞周期调控的影响

By homo-recombination with yeast intergrating plasmids, a serial of haploid mutants of budding yeast YPH499 had been constructed, which included pho85 delta strain YPH600, pho85 delta cln1 delta strain YPH610, cln1 delta cln2 delta strain YPH640 and galactose inducible strain YPH630 (pho85 delta cln1 delta cln2 delta (GAL1-10PHO85)). By analyzing the growth rate of different strains, we concluded that PHO85 gene have greater influence than CLN1 and CLN2 genes on cell growth control. After transferred from galactose media to glucose media, the tri-mutant cells collected at intervals were observed with microscope and analyzed by FACS. The results showed that the tri-mutant cells arrest in G1 phase when they were transferred to glucose media.     

Mutations in RAD27 define a potential link between G1 cyclins and DNA replication.

The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions. To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2. We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2. rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to a DNA-damaging agent, increased mitotic recombination rates, and increased spontaneous mutation rates. Unlike the block in cell cycle progression, these phenotypes are not suppressed by CLN1 or CLN2. CLN1 and CLN2 may activate an RAD27/ERC11-independent pathway specific for DNA synthesis that CLN3 is incapable of activating. Alternatively, CLN1 and CLN2 may be capable of overriding a checkpoint response which otherwise causes cln1 cln2 rad27/erc11 cells to arrest. These results imply that CLN1 and CLN2 have a role in the regulation of DNA replication. Consistent with this, GAL-CLN1 expression in checkpoint-deficient, mec1-1 mutant cells results in both cell death and increased chromosome loss among survivors, suggesting that CLN1 overexpression either activates defective DNA replication or leads to insensitivity to DNA damage.     

C1n3缺失对酿酒酵母生长的影响

Cln3是酿酒酵母G1期周期蛋白中的一种,为了研究Cln3在细胞周期与形态发生中的作用,我们构建了酿酒酵母CLN3基因的缺失株,并对其表型进行了分析。结果显示,cln3缺失株对α信息素的敏感性增强,α信息素诱导的细胞周期停滞现象明显大于野生型菌株,这种增强作用不受Sgvl因子的影响。同时,与野生菌相比cln3缺失株的细胞形态也有明显变化,双倍体cln3缺失株细胞的顶端生长能力增强而单倍体细胞的侵入生长能力则受到抑制。结果表明,与酿酒酵母的另外两个G1期周期蛋白Clnl、Cln2不同,Cln3在形态发生中有其独特的功能与作用方式。     

A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle

The CLN1, CLN2, and CLN3 genes of S. cerevisiae form a redundant family essential for the G1-to-S phase transition. CLN1 and CLN2 mRNAs were previously shown to be negatively regulated by mating pheromone and by cell cycle progression out of G1, whereas CLN3 mRNA is not. The CLN3-2 (DAF1-1) allele prevents both cell cycle arrest and the turnoff of CLN1 and CLN2 mRNAs in response to mating pheromone, but only in the presence of an active CDC28 gene. An internally deleted nonfunctional cln2 gene was used as a reporter gene to demonstrate that in the absence of mating pheromone, efficient expression of cln2 mRNA requires both an active CDC28 gene and at least one functional CLN gene. mRNA from a nonfunctional cln1 gene was regulated similarly. Thus, CLN function and CDC28 activity jointly stimulate CLN1 and CLN2 mRNA levels, potentially forming a positive feedback loop for CLN1 and CLN2 expression.     

Cln3 function is linked to osmoregulation in a Dictyostelium model of Batten disease

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease. Currently, there is no cure for NCL and the mechanisms underlying the disease are not well understood. In the social amoeba Dictyostelium discoideum, the CLN3 homolog, Cln3, localizes predominantly to the contractile vacuole (CV) system. This dynamic organelle functions in osmoregulation, and intriguingly, osmoregulatory defects have been observed in mammalian cell models of CLN3 disease. Therefore, we used Dictyostelium to further study the involvement of CLN3 in this conserved cellular process. First, we assessed the localization of GFP-Cln3 during mitosis and cytokinesis, where CV system function is essential. GFP-Cln3 localized to the CV system during mitosis and cln3^? cells displayed defects in cytokinesis. The recovery of cln3^? cells from hypotonic stress and their progression through multicellular development was delayed and these effects were exaggerated when cells were treated with ammonium chloride. In addition, Cln3-deficiency reduced the viability of cells during hypotonic stress and impaired the integrity of spores. During hypertonic stress, Cln3-deficiency reduced cell viability and inhibited development. We then performed RNA sequencing to gain insight into the molecular pathways underlying the sensitivity of cln3^? cells to osmotic stress. This analysis revealed that cln3-deficiency upregulated the expression of tpp1A, the Dictyostelium homolog of human TPP1/CLN2. We used this information to show a correlated increase in Tpp1 enzymatic activity in cln3^? cells. In total, our study provides new insight in the mechanisms underlying the role of CLN3 in osmoregulation and neurodegeneration.     

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.