酵母PHO2蛋白及其变异体与PHO5USA体外的相互作用

酵母ＰＨＯ２蛋白及其变异体与ＰＨＯ５ＵＳＡ体外的相互作用杨军,敖世洲（中国科学院上海生物化学研究所分子生物学国家重点实验室,２０００３１）关键词酵母;ＰＨＯ２;突变;ＤＮＡ结合ＰＨＯ２是酵母阻遏型酸性磷酸酯酶基因转录的正调控因子［１］,由５５９个氨基...     

PHO2-dependent degradation of PHO1 modulates phosphate homeostasis in Arabidopsis

The Arabidopsis thaliana pho2 mutant, which is defective in a ubiquitin-conjugating E2 enzyme, displays inorganic phosphate (Pi) toxicity as a result of enhanced uptake and root-to-shoot translocation of Pi. To elucidate downstream components of the PHO2-dependent regulatory pathway, we identified two pho2 suppressors as carrying missense mutations in PHO1, which has been implicated in Pi loading to the xylem. In support of the genetic interaction between PHO1 and PHO2, we found that the protein level of PHO1 is increased in pho2, whereas such accumulation is ameliorated in both pho2 suppressors. Results from cycloheximide and endosomal Cys protease inhibitor E-64d treatments further suggest that PHO1 degradation is PHO2 dependent and involves multivesicular body-mediated vacuolar proteolysis. Using the transient expression system of tobacco (Nicotiana tabacum) leaves, we demonstrated that PHO1 and PHO2 are partially colocalized and physically interact in the endomembranes, where the ubiquitin conjugase activity of PHO2 is required for PHO1 degradation. In addition, reduced PHO1 expression caused by PHO1 mutations impede Pi uptake, indicating a functional association between xylem loading and acquisition of Pi. Together, our findings uncover a pivotal molecular mechanism by which PHO2 modulates the degradation of PHO1 in the endomembranes to maintain Pi homeostasis in plants.     

The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2). Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2). Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites. DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.     

PHO80、PHO85基因和芽殖酵母金属离子耐受

PHO8 5基因是芽殖酵母中的一个多功能基因。它参与了无机磷酸的代谢、碳源利用、糖原积累、特定蛋白质的降解和细胞周期调控。研究了酵母株YPH499及其衍生的pho85缺失株、pho80缺失株、pap1(pcl7)缺失株在不同浓度的不同金属离子中的存活情况 ,结果表明和芽殖酵母YPH499相比 ,pho85缺失株和pho80缺失株表现出对K 、Mg2 、Zn2 、Ca2 和Mn2 的耐受下降 ,而PAP1基因的缺失则不会导致芽殖酵母对上述金属离子的敏感性的变化 ;而对Cu2 ,3株突变株都表现出和YPH499相同的耐受性。同时测定了各缺失株和YPH499对上述金属离子的半致死浓度以及pho85缺失株、pho80缺失株和YPH499的细胞内总钙量。这些结果显示 ,PHO85蛋白激酶通过和它的PCLPHO80而不是PAP1结合 ,参与了芽殖酵母K 、Mg2 、Zn2 、Ca2 和Mn2 离子平衡的调控。PHO85和PHO80基因的缺失损害了芽殖酵母钙的储存。     

Negative regulators of the PHO system in Saccharomyces cerevisiae: isolation and structural characterization of PHO85.

One of the negative regulators of the PHO system of Saccharomyces cerevisiae, PHO85, has been isolated by transformation and complementation of a pho85 strain. The complementing activity was delimited within a 1258 bp DNA segment and this region has been sequenced. The largest open reading frame found in this region can encode a protein of 302 amino acid residues. A pho85 mutant resulted from disruption of the chromosomal counterpart of the open reading frame described above. Therefore, we concluded that the gene we have cloned is PHO85. This result also indicates that PHO85 is nonessential. Northern analysis revealed that the size of the PHO85 message is 1.1 kb. No similarity was found between the putative amino acid sequences of two negative regulators, the PHO80 and PHO85 proteins.     

酵母PHO2与PHO4蛋白的激活活性的分析及两者的相互作用

ＰＨＯ２与ＰＨＯ４是酵母ＰＨＯ５基因的两个正调控因子,本文发现,ＰＨＯ２与酵母转录因子ＧＡＬ４的ＤＮＡ结合功能域融合后就能激活报道基因ｌａｃＺ的表达,其激活力受高低磷影响,表明ＰＨＯ２蛋白上存在酸性转录激活区。ＰＨＯ２蛋白上酸性氨基酸丰富的２８７－３２６肽段并非ＰＨＯ２的激活区。在ＰＨＯ２蛋白上２３０位Ｓｅｒ处于磷酸化状态２ＰＨＯ２才有激活作用,表明了这一磷酸化位点可能与ＰＨＯ２的转录激活能力有关     

Nucleosome disruption at the yeast PHO5 promoter upon PHO5 induction occurs in the absence of DNA replication.

Activation of the PHO5 gene in S. cerevisiae by phosphate starvation was previously shown to be accompanied by the disappearance of four positioned nucleosomes from the promoter. To investigate the mechanism, we replaced the PHO80 gene, a negative regulator of PHO5, by a temperature-sensitive allele. As a consequence, PHO5 can be activated in the presence of phosphate by a temperature shift from 24 degrees C to 37 degrees C. Under these conditions, the promoter undergoes the same chromatin transition as in phosphate-starved cells. Disruption of the nucleosomes by the temperature shift also occurs when DNA replication is prevented. Nucleosomes re-form when the temperature is shifted from 37 degrees C back to 24 degrees C in nondividing cells. Glucose is required for the disruption of the nucleosomes during the temperature upshift, not for their re-formation during the temperature downshift. These experiments prove that DNA replication is not required for the transition between the nucleosomal and the non-nucleosomal state at the PHO5 promoter.     

The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters

Eukaryotic gene expression starts off from a largely obstructive chromatin substrate that has to be rendered accessible by regulated mechanisms of chromatin remodeling. The yeast PHO5 promoter is a well known example for the contribution of positioned nucleosomes to gene repression and for extensive chromatin remodeling in the course of gene induction. Recently, the mechanism of this remodeling process was shown to lead to the disassembly of promoter nucleosomes and the eviction of the constituent histones in trans. This finding called for a histone acceptor in trans and thus made histone chaperones likely to be involved in this process. In this study we have shown that the histone chaperone Asf1 increases the rate of histone eviction at the PHO5 promoter. In the absence of Asf1 histone eviction is delayed, but the final outcome of the chromatin transition is not affected. The same is true for the coregulated PHO8 promoter where induction also leads to histone eviction and where the rate of histone loss is reduced in asf1 strains as well, although less severely. Importantly, the final extent of chromatin remodeling is not affected. We have also presented evidence that Asf1 and the SWI/SNF chromatin remodeling complex work in distinct parallel but functionally overlapping pathways, i.e. they both contribute toward the same outcome without being mutually strictly dependent.     

酵母PHO4基因的克隆与表达

用原位杂交方法从酵母菌染色体ＤＮＡ中分段克隆,拼接,获得完整的ＰＨＯ４基因,全长约３．４ｋｂ,利用体内同源重组的方法,构建了ＰＨＯ４基因缺失突变株。ＰＨＯ４基因的缺失导致酸性磷酸酯酶活性明显下降,将完整的ＰＨＯ４基因转入这种缺陷细胞,能使酶活性回复到野生型水平,ＰＨＯ４起正调控作用,构建ＰＨＯ４－ＬａｃＺ融合基因,以β－半乳糖苷酶的活力表示ＰＨＯ４的表达水平。融合基因不同名的表达表明,ＰＨＯ４基因     

Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements.

The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.     

Nucleosome stability at the yeast PHO5 and PHO8 promoters correlates with differential cofactor requirements for chromatin opening

The coregulated PHO5 and PHO8 genes in Saccharomyces cerevisiae provide typical examples for the role of chromatin in promoter regulation. It has been a long-standing question why the cofactors Snf2 and Gcn5 are essential for full induction of PHO8 but dispensable for opening of the PHO5 promoter. We show that this discrepancy may result from different stabilities of the two promoter chromatin structures. To test this hypothesis, we used our recently established yeast extract in vitro chromatin assembly system, which generates the characteristic PHO5 promoter chromatin. Here we show that this system also assembles the native PHO8 promoter nucleosome pattern. Remarkably, the positioning information for both native patterns is specific to the yeast extract. Salt gradient dialysis or Drosophila embryo extract does not support proper nucleosome positioning unless supplemented with yeast extract. By competitive assemblies in the yeast extract system we show that the PHO8 promoter has greater nucleosome positioning power and that the properly positioned nucleosomes are more stable than those at the PHO5 promoter. Thus we provide evidence for the correlation of inherently more stable chromatin with stricter cofactor requirements.     

Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast.

The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays.