酵母蛋白质YJL084c的磷酸化分析

PCL6、PCL7(PAP1)和PHO80为酵母蛋白激酶PHO85的细胞周期蛋白因子 ,同属于PHO80亚类 ,它们在蛋白质序列上有较高的同源性 ,彼此在功能上有一定程度的重叠。YLR190w为先前鉴定的PCL7 PHO85的一个底物 ,根据BLAST比较 ,YJL0 84c与YJLR190w在一段区域内有一定的同源性 ,并且根据已公布公共数据库 ,YJL0 84c是PCL6的一个结合蛋白。利用双杂交系统分析了YJL0 84c与这三个细胞周期蛋白因子之间的相互作用 ,表明PCL7也可以结合YJL0 84c。利用免疫共沉淀证实它们在体内的相互作用 ;通过体外GST沉降分析 ,验证它们的体外相互作用。YJL0 84c的体外翻译产物可以被PCL7 PHO85复合物磷酸化 ,进一步分别表达纯化YJL0 84c的氨基末端、中间和羧端三部分的GST融合蛋白片段 ,PCL7 PHO85复合物可以磷酸化其中间部分 ,而对N末端和C末端则未表现出磷酸化。以该中间片段蛋白质为底物 ,进一步研究高低磷条件对其磷酸化的影响。PCL7 PHO85对YJL0 84c的磷酸化受到无机磷条件的明显影响 :高磷条件下 ,磷酸化程度高 ;而低磷条件下磷酸化程度低。有意思的是 ,PCL6 PHO85免疫沉淀复合物也表现出同样的磷酸激酶特征。于是分析PHO81与这三个细胞周期因子之间相互作用。此外 ,还构建了Yjl0 84c的缺失株 ,分析了缺失的可能影响。     

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。     

鉴定人甲基化CpG结合结构域蛋白4为蛋白激酶X的底物

人蛋白激酶X(PrKX)是由X染色体编码的一种cAMP依赖性蛋白激酶,但是到目前为止已鉴定到的PrKX底物还很少.为了鉴定蛋白激酶X的底物,我们以蛋白激酶X为诱饵进行了酵母双杂交实验,结果发现甲基化CpG结合结构域蛋白4(MBD4)与PrKX在酵母细胞内相互作用较强.GST融合蛋白沉降和细胞内蛋白质的免疫共沉淀证实PrKX与MBD4之间确实存在相互作用.进一步研究表明,大肠杆菌中表达的重组MBD4在体外可以被PrKX磷酸化,而且MBD4蛋白的磷酸化能明显增强它在体外与甲基化DNA探针的结合活性.     

细胞周期负调控

调控细胞周期的关键是调节细胞周期蛋白依赖性蛋白激酶(CDK)的活性。细胞周期蛋白可结合并激活CDK,CDK活性还可通过磷酸化作用调节。因此细胞周期负调控包括以下3点:①细胞周期蛋白降解速度;②CDK磷酸化状态;③CDK抑制蛋白(CKI)。酵母中CKI包括FAR1,p40、PHO81,哺乳动物CKI有p21家族(包括p21、p27)及p16家族(包括p16、p15)。细胞周期负调控与抑癌基因密切相关,是不同抗肿瘤因子作用的共同途径。     

酵母Pho80对Pho85蛋白激酶活性的调节

酵母pho80和pho85基因编码的蛋白质是阻遏型酸性磷酸酯酶基因表达调控系统中的2种负调控因子.其中pho85基因编码产物己被证明是1种类似于p34~(^cdc2/CDC28)的蛋白激酶,磷酸化该调控系统中的正调控因子Pho4,并使之失活,用抗pho85抗体从啤酒酵母YPH499及其衍生菌株的细胞抽提液中得到Pho85免疫复合物,对大肠杆菌表达的Pho4蛋白进行了体外磷酸化分析,证实酵母Pho80是Pho85蛋白激酶活力所必需,pho80基因的表达水平直接影响Pho85免疫复合物对Pho4蛋白的磷酸化程度.根据基因序列推导的Pho80蛋白氨基酸序列中,含有一段与几种cyclin同源的区域,通过寡核苷酸的插入或小片段缺失而对该区域及邻近部位在基因水平进行的微小改变均可导致Pho80丧失阻遏酸性磷酸酯酶基因表达的能力.     

HTLV—1转录激活因子Tax和Taxreb107的相互作用

Tax是人类T淋巴细胞白血病病毒编码的转录因子。核糖体蛋白RpL6又称Taxreb10 7,是Tax应答序列结合蛋白 ,二者均作用于HTLV 1的启动子LTR。用酵母双杂交法和GST下拉检测 (pull down)法研究了Tax和Taxreb10 7/RpL6之间的相互关系。结果显示 ,二者在酵母细胞内和体外均具有直接相互作用。这些结果提示Taxreb10 7/RpL6可能通过与Tax的相互作用而调节Tax在病毒感染中的作用     

ORP8 与SPAG5 相互作用并影响细胞周期

目的:筛选与氧化固醇结合蛋白相关蛋白8(Oyxterol binding protein related protein 8,ORP8)相互作用的蛋白质,为揭示ORP8在细胞中的功能及其机制提供线索,并根据相互作用蛋白质初步探索ORP8的功能.方法:构建重组诱饵质粒Pgbkt7-ORP8m,利用GAL4酵母双杂交系统筛选人Universal Cdna文库,筛选出与ORP8相互作用的蛋白质,通过GST Pull-down以及免疫共沉淀(Co-immunoprecipitation,Co-IP)验证蛋白质相互作用,并通过流式细胞仪检测过表达ORP8对HepG2细胞周期的影响.结果:酵母双杂交筛选得到了精子相关抗原5(Homo sapiens sperm associated antigen 5,SPAG5),体外GST pull-down和Co-IP实验确证了ORP8-SPAG5的相互作用,流式细胞术显示ORP8过表达后与空载体相比,人肝癌细胞系HepG2的S期细胞数增加,G1期细胞数减少.结论:SPAG5是与ORP8相互作用的蛋白质,ORP8过表达影响人肝癌细胞系HepG2的细胞周期,可能通过SPAG5起作用.ORP8-SPAG5的相互作用为进一步研究ORP8在肝细胞中的功能及其机制提供了有用的线索     

LIM蛋白KyoT与PcG蛋白的相互作用

KyoT为LIM结构域蛋白 ,可与转录因子RBP J相互作用并抑制RBP J介导的转录激活。为了研究其功能 ,用酵母双杂交法筛选了与KyoT相互作用的蛋白质 ,其中包括同属于PcG (polycombgroup)家族的蛋白质Ring1和hPc2 (humanpolycomb2 )。为进一步验证KyoT2与Ring1和hPc2的相互作用 ,首先在酵母中证实了KyoT2与它们的相互作用并得到阳性结果。然后做GST pulldown实验在体外验证了KyoT2与Ring1和hPc2在体外的相互作用。进而又构建了KyoT2与Ring1和hPc2不同的截短体 ,利用酵母双杂交进一步验证了它们相互作用的区段 ,发现KyoT的LIM结构域与Ring1的全长相互作用 ,与hPc2的C末端和全长相互作用。本研究验证了KyoT2与Ring1和hPc2的相互作用 ,对探讨KyoT的功能以及分析Notch信号途径与PcG家族的关系提供一定的依据。     

氧化葡萄糖酸杆菌中一种潜在的双组分系统蛋白的功能研究

氧化葡萄糖酸杆菌(Gluconobacteroxydans)基因组编码的蛋白质中,有相当数量的传感器激酶和反应调控蛋白组成了细菌的多个双组分信号转导系统(two-componentsignaltransduction systems, TCSs),这些系统能够介导细菌对外界环境变化做出反应。但目前对G. oxydans中潜在的双组分系统成员蛋白质结构和功能缺少必要的研究【。目的】研究菌株G. oxydans 621H中GOX0645基因序列所编码蛋白质的自磷酸化活性,探究其与细菌趋化性运动的关联,揭示其是否作为一种双组分系统成员蛋白在细胞内发挥作用。【方法】以菌株G. oxydans 621H基因组中一段可能编码双组分系统蛋白质的基因GOX0645为基础,通过生物信息学分析其保守结构域;采用体外化学发光实验证明其编码蛋白的自磷酸化活性;利用基因定点突变筛选出与自磷酸化活性相关的氨基酸位点;通过差速离心法寻找双组分蛋白的亚细胞定位;最后运用体内双分子荧光互补和体外生物大分子相互作用实验印证其与下游鞭毛马达蛋白之间的相互作用。【结果】生物信息学分析发现GOX0645编码蛋白同时具有组氨酸激...     

CPSF蛋白相互作用的体外研究

目的：研究在前体mRNA的切割与多聚腺嘌呤化中4种CPSF蛋白间的相互作用及CPSF73k、100k、160k与PAP的相互作用。方法：将GST—CPSF30k和GST—PAP结合于谷胱甘肽Sepharose基质上，与经网织红细胞体外翻译的CPSF73k、100k、160k蛋白进行GST—pUll down试验。结果与讨论：蛋白质电泳结果表明，4种CPSF蛋白可相互作用，以四聚体的形式与参与前体mR—NA3’端加工。此外，CPSF73k、100k、160k也可分别与PAP相互作用，共同完成前体mRNA的切割与多聚腺嘌呤化。     

Pex30p, Pex31p, and Pex32p form a family of peroxisomal integral membrane proteins regulating peroxisome size and number in Saccharomyces cerevisiae

The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome. Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size. Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes. Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p. Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number. Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation. Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p--now designated Pex30p, Pex31p, and Pex32p, respectively--together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.     

Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component

In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1.     

Pin1 modulates the dephosphorylation of the RNA polymerase II C-terminal domain by yeast Fcp1

The reversible phosphorylation of serine and threonine residues N-terminal to proline (pSer/Thr-Pro) is an important signaling mechanism in the cell. The pSer/Thr-Pro moiety exists in the two distinct cis and trans conformations, whose conversion is catalyzed by the peptidyl-prolyl isomerase (PPIase) Pin1. Among others, Pin1 binds to the phosphorylated C-terminal domain (CTD) of the largest subunit of the RNA polymerase II, but the biochemical and functional relevance of this interaction is unknown. Here we confirm that the CTD phosphatase Fcp1 can suppress a Pin1 mutation in yeast. Furthermore, this genetic interaction requires the phosphatase domain as well as the BRCT domain of Fcp1, suggesting a critical role of the Fcp1 localization. Based on these observations, we developed a new in vitro assay to analyze the CTD dephosphorylation by Fcp1 that uses only recombinant proteins and mimics the in vivo situation. This assay allows us to present strong evidence that Pin1 is able to stimulate CTD dephosphorylation by Fcp1 in vitro, and that this stimulation depends on Pin1's PPIase activity. Finally, Pin1 significantly increased the dephosphorylation of the CTD on the Ser(5)-Pro motif, but not on Ser(2)-Pro in yeast, which can be explained with Pin1's substrate specificity. Together, our results indicate a new role for Pin1 in the regulation of CTD phosphorylation and present a further example for prolyl isomerization-dependent protein dephosphorylation.     

Effect of the Phosphate Group with Different Negative Charges on the Conformation of Phosphorylated Ser/Thr-Pro Motif

Summary Ser/Thr-Pro motif is a widespread phosphorylated site in proteins, and its reversible phosphorylation is an important regulatory progress in many cell cycles and signal transduction. Recent research reveals that phosphorylation affects the local conformation of the peptide and its binding with the substrate through peptidyl--prolyl cis/trans isomerization. In order to further explore the effect of the phosphate group with different charges, four model peptides containing non- and phosphorylated Ser/Thr-Pro motif were synthesized using the classical solid-phase method. 1H-NMR, TOCSY, and ROESY were employed to characterize the conformation of the model peptides in solution with different pH value and analyze the peptidyl--prolyl isomerization at a molecular level. The results demonstrate that phosphorylation increases the cis conformation in the peptide and the maximum cis/trans ratio is obtained when the phosphate group has two negative charges. Furthermore, the experiments prove that the phosphorylation introduces a hydrogen bond between the phosphate and the NH of Ser/Thr residue, and the charges of the phosphate affect certain conformations of the phosphorylated Ser/Thr-Pro motif.     

Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II and III

Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure. They all impair a two-hybrid interaction with a fragment conserved in the largest subunits of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p). This fragment corresponds to the pore 1 module of the RNA polymerase II crystal structure and bears a highly conserved motif (P.I.KP.LW.GKQ) facing the GGLLM motif of Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP.LW.GKQ provokes a temperature-sensitive defect. Increasing the gene dosage of another common subunit, Rpb6p, suppresses this phenotype. It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart. Hence, Rpb6p and Rpb8p functionally interact in vivo. These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-Delta null allele. A two-hybrid interaction also occurs between Rpb8p and the product of orphan gene YGR089w. A ygr089-Delta null mutant has no detectable growth defect but aggravates the conditional growth defect of rpb8 mutants, suggesting that the interaction with Rpb8p may be physiologically relevant.     

A genomic screen for yeast mutants defective in mitophagy

Mitochondria autophagy (mitophagy) is the process of selective degradation of mitochondria that has an important role in mitochondrial quality control. To gain insight into the molecular mechanism of mitophagy, we screened a yeast knockout library for strains that are defective in mitophagy. We found 32 strains that showed a complete or partial block of mitophagy. One of the genes identified, YLR356W, is required for mitophagy, but not for macroautophagy or other types of selective autophagy. The deletion of YLR356W partially inhibits mitophagy during starvation, whereas there is almost complete inhibition at post-log phase. Accordingly, we hypothesize that Ylr356w is required to detect or present aged or dysfunctional mitochondria when cells reach the post-log phase.     

Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.