Glucose Monitoring in Fission Yeast via the gpa2 Ga, the git5 Gb and the git3 Putative Glucose Receptor
Glucose Monitoring in Fission Yeast via the gpa2 Ga, the git5 Gb and the git3 Putative Glucose Receptor
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通过 gpa2 Ga、git5 Gb 和 git3 假定葡萄糖受体监测裂殖酵母中的葡萄糖
DOI：
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
C. S. Hoffman
中科院分区：
文献类型：
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作者：
R. Welton;C. S. Hoffman
分裂酵母裂糖酵母pombe通过激活腺苷酸环化酶对环境葡萄糖作出反应。由此产生的cAMP信号激活蛋白激酶A （PKA）。PKA抑制葡萄糖饥饿诱导的过程，如偶联和减数分裂，以及编码糖异生酶果糖-1,6-二磷酸酶的fbp1基因的转录。我们之前确定了葡萄糖抑制fbp1转录所需的git基因集合，包括编码PKA催化亚基的pka1/git6，编码腺苷酸环化酶的git2/cyr1，以及腺苷酸环化酶激活所需的六个“上游”基因。git8基因与gpa2相同，编码异三聚体鸟嘌呤核苷酸结合蛋白（Ga）的α亚基，而git5基因编码Gb亚基。先前对gpa2的多拷贝抑制研究表明，S. pombe腺苷酸环化酶的激活可能类似于哺乳动物II型酶的顺序激活，先被Ga激活，再被Gbg激活。我们在这里展示了一个激活的gpa2等位基因（gpa2，在GTPase结构域的编码区携带一个突变）完全抑制git3和git5的突变，导致我们模型的改进。我们描述了git3的克隆，并表明它编码一个假定的七跨膜G蛋白偶联受体。git3缺失与PKA通路的其他组分缺失具有相同的表型，包括发芽延迟、构成性fbp1转录和不依赖饥饿的偶联。由于gpa2 R176H等位基因在fbp1转录中完全抑制git3的缺失，git3似乎编码了一个G蛋白偶联的葡萄糖受体，负责S. pombe中腺苷酸环化酶的激活。环境葡萄糖是一个重要的调节机制，酿酒酵母中的葡萄糖检测通过多种机制发生，这些机制在单细胞生物和哺乳动物中仍在积极地研究基因表达和其他生物过程。一种由RGT2和SNF3编码的葡萄糖传感器类似于一个12-跨膜己糖跨细胞。因此，大量的研究已经投入到波特（Ozcan和Johnston 1999年评论）。A对葡萄糖检测的研究和相关的第二葡萄糖检测系统负责多种模式有机酸环化酶激活的信号转导途径。GPR1和GPA2基因，也就是isms。这些研究揭示了编码假定的七跨膜蛋白的惊人差异，以及两个关键模型系统，细菌异三聚体G蛋白α亚基（Ga），大肠杆菌和出芽酵母酵母是葡萄糖检测酵母的关键成分，如何检测和响应葡萄糖。途径(Colombo et al. 1998; Xue et al. 1998； Yun等大肠杆菌采用依赖磷酸烯醇丙酮酸的磷。1998年;Kraakman et al. 1999；Lorenz et al. 2000)。虽然gpa2ga似乎不与葡萄糖感知及其易位和光经典Gbg二聚体相互作用，但尚不清楚它是否磷酸化葡萄糖-6-磷酸（Postma将其作为单体或在其他蛋白质复合物中进行审查）。Et al. 1993)。因此，大肠杆菌中的葡萄糖检测是Gpr1和Gpa2也涉及到与其摄取内在相关的控制。假菌丝生长的葡萄糖PTS系统（Lorenz and Heitman 1997；通过抑制ansari等人，1999;Pan and Heitman 1999）调节替代碳源的利用，关键是其他碳源的运输（Mep2渗透酶作为氨排除的诱导剂作用），并通过降低腺苷酸环化酶活性，monium传感器和Gpr1作为碳源传感器，从而降低细胞内cAMP水平。减少（Lorenz and Heitman 1998; Lorenz et al. 2000）。分裂酵母裂糖酵母（Schizosaccharomyces pombe）监测cAMP受体蛋白，这是一种积极的转录葡萄糖调节剂，可以调节广泛的生物过程。受葡萄糖抑制的操纵子的数目。我们的研究重点是葡萄糖抑制fbp1基因的转录调控，该基因编码糖异生酶果糖-1,6-二磷酸酶（vassar通讯作者：Charles S. Hoffman, Department of biological, otti and Friesen 1985）。以前，我们确定了波士顿学院，希金斯厅401B，栗子山，MA 02467。E-mail: hoffmacs@bc.edu基因中给予构成性fbp1转录的基因156:513-521（2000年10月）514 R. M. Welton和C. S. Hoffman （Hoffman和Winston 1990）。这些git（葡萄糖失活git3并提供遗传证据，证明它编码负责敏感转录的G蛋白偶联受体）基因在PKA途径中起作用（Hoffman and Winston 1991； Byrne and Hoffman通过gpa2激活腺苷酸环化酶）。1993)。git2基因与cyr1相同（YamawakiKataoka et al. 1989; Young et al. 1989; Maeda et al.）。材料和方法1990)，编码腺苷酸环化酶（Hoffman和Winston 1991）；git6基因，与pka1相同（Maeda et Yeast菌株和本研究使用的生长介质：S. pombe菌株）见表1。fbp1::ura4等位基因是一个干扰（disruption al. 1994），编码PKA的催化亚基（Jin等人通过ura4基因的编码区编码fbp1基因，creating al. 1995）。其余6个基因，git1, git3, git5, git7，在转录控制下的翻译融合git8和git10，是葡萄糖触发fbp1启动子的adef所必需的。ura4::fbp1-lacZ等位基因是一个中断nylate环化酶激活。通过fbp1-lacZ翻译融合在这些ura4基因的“上游”发生突变（Hoffman和Winston 1990）。git基因被多拷贝git2或外源标准富培养基YEA和YEL （Gutz et . 1974）抑制，而cAMP （Hoffman and Winston 1991）和添加2% casamino acids的菌株则被抑制。PM培养基（Watanabe携带这些基因中的任何一个突变都无法升高等人，1988年）在葡萄糖（Byrne mg/l，亮氨酸为150 mg/l）的作用下，以75 mg/l的细胞内cAMP水平补充所需的营养素。Gluand Hoffman 1993)。git8基因与gpa2相同，除非另有说明，否则以3%的浓度存在。对5-氟乙酸（5-FOA）的敏感性以Ga亚基来测定（Isshiki等人，1992年；Nocero等人在含有0.4 g/l 5-FOA的SC固体培养基上进行了测定，1994年）。如前所述，多拷贝gpa2部分抑制muta8%葡萄糖（Hoffman和Winston在git3和git5中发现，但在其他上游git5中没有发现）。菌株在308℃下生长。对基因进行杂交（Nocero et al. 1994; Landry et al. 2000）。SPA （Gutz et al. 1974）在PM培养基上进行预生长。git5基因编码一个Gb亚基，作为一个阳性上位性测试：上位性测试是通过检测gpa2 Ga的RWP4调节子杂交的四分体分离后代来进行的（Landry et al. 2000）。pka1/git6、git7和git10的遗传特征在这篇文章中，我们进一步描述了git5、pka1/git6、git7和git10的遗传特征。在gpa2萌发并与其他上游基因形成相互作用后，将后代转移到新鲜的YEA板上，基因通过使用gpa2的“激活”等位基因生长1天，然后复制镀到含有其产物在其自抑制GTPase培养基中存在缺陷的5- foa上。复镀后2-3天测定5-FOA抗性。活动。此外，我们描述了克隆和char-
The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvationinduced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six “upstream” genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guaninenucleotide binding protein (Ga) while git5 encodes a Gb subunit. Multicopy suppression studies with gpa2 previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Ga followed by Gbg. We show here that an activated allele of gpa2 (gpa2 , carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2 R176H allele with respect to fbp1 transcription, git3 appears to encode a G proteincoupled glucose receptor responsible for adenylate cyclase activation in S. pombe. ENVIRONMENTAL glucose is an important regulaGlucose detection in S. cerevisiae occurs through multiple mechanisms that are still actively under examinator of gene expression and other biological processes in both unicellular organisms and mammalian tion. One type of glucose sensor, encoded by RGT2 and SNF3, resembles a 12-transmembrane hexose transcells. As such, considerable research has been devoted porter (reviewed by Ozcan and Johnston 1999). A to the study of glucose detection and the associated second glucose detection system is responsible for adesignal transduction pathways in a variety of model organnylate cyclase activation. The GPR1 and GPA2 genes, isms. These studies have revealed surprising differences encoding a putative seven-transmembrane protein and with respect to how two key model systems, the bactea heterotrimeric G protein alpha subunit (Ga), respecrium Escherichia coli and the budding yeast Saccharomyces tively, are key components in this glucose-detection cerevisiae, detect and respond to glucose. pathway (Colombo et al. 1998; Xue et al. 1998; Yun et E. coli employs a phosphoenolpyruvate-dependent phosal. 1998; Kraakman et al. 1999; Lorenz et al. 2000). photransferase system (PTS) that is responsible for both While the Gpa2 Ga does not appear to interact with a the sensing of glucose and its translocation and phosclassical Gbg dimer, it is unclear whether it functions phorylation to glucose-6-phosphate (reviewed by Postma as a monomer or within some other protein complex. et al. 1993). Therefore, glucose detection in E. coli is Gpr1 and Gpa2 have also been implicated in the control intrinsically linked to its uptake. The glucose PTS system of pseudohyphal growth (Lorenz and Heitman 1997; regulates alternative carbon source utilization by inhibAnsari et al. 1999; Pan and Heitman 1999), with key iting the transport of other carbon sources (inducer roles postulated for both the Mep2 permease as an amexclusion) and by reducing adenylate cyclase activity, monium sensor and Gpr1 as a carbon source sensor thus lowering intracellular cAMP levels. The reduced (Lorenz and Heitman 1998; Lorenz et al. 2000). cAMP level causes a reduction in DNA binding by the The fission yeast Schizosaccharomyces pombe monitors cAMP receptor protein, a positive regulator of transcripglucose to regulate a wide range of biological processes. tion of operons subject to glucose repression. Our studies focus on the transcriptional regulation of the glucose-repressed fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase (VassarCorresponding author: Charles S. Hoffman, Department of Biology, otti and Friesen 1985). Previously, we identified mutaBoston College, Higgins Hall 401B, Chestnut Hill, MA 02467. E-mail: hoffmacs@bc.edu tions in genes that confer constitutive fbp1 transcription Genetics 156: 513–521 (October 2000) 514 R. M. Welton and C. S. Hoffman (Hoffman and Winston 1990). These git (glucose inacterization of git3 and provide genetic evidence that it encodes the G protein-coupled receptor responsible for sensitive transcription) genes act in a PKA pathway (Hoffman and Winston 1991; Byrne and Hoffman the activation of adenylate cyclase through gpa2. 1993). The git2 gene, identical to cyr1 (YamawakiKataoka et al. 1989; Young et al. 1989; Maeda et al. MATERIALS AND METHODS 1990), encodes adenylate cyclase (Hoffman and Winston 1991); the git6 gene, identical to pka1 (Maeda et Yeast strains and growth media: S. pombe strains used in this study are listed in Table 1. The fbp1::ura4 allele is a disruption al. 1994), encodes the catalytic subunit of PKA (Jin et of the fbp1 gene by the coding region of the ura4 gene, creating al. 1995). The remaining six genes, git1, git3, git5, git7, a translational fusion that is under the transcriptional control git8, and git10, are required for glucose-triggered adeof the fbp1 promoter. The ura4::fbp1-lacZ allele is a disruption nylate cyclase activation. Mutations in these “upstream” of the ura4 gene by an fbp1-lacZ translational fusion (Hoffman and Winston 1990). git genes are suppressed by multicopy git2 or by exogeStandard rich media YEA and YEL (Gutz et al. 1974) were nous cAMP (Hoffman and Winston 1991), and strains supplemented with 2% casamino acids. PM media (Watanabe carrying mutations in any of these genes fail to elevate et al. 1988) were supplemented with required nutrients at 75 intracellular cAMP levels in response to glucose (Byrne mg/liter, except for leucine which was at 150 mg/liter. Gluand Hoffman 1993). The git8 gene, identical to gpa2, cose was present at a concentration of 3%, unless otherwise specified. Sensitivity to 5-fluoro-orotic acid (5-FOA) was deterencodes a Ga subunit (Isshiki et al. 1992; Nocero et mined on SC solid medium containing 0.4 g/liter 5-FOA and al. 1994). Multicopy gpa2 partially suppresses muta8% glucose as previously described (Hoffman and Winston tions in git3 and git5 but not in the other upstream git 1990). Strains were grown at 308. Crosses were performed on genes (Nocero et al. 1994; Landry et al. 2000). The SPA (Gutz et al. 1974) following pregrowth on PM medium. git5 gene encodes a Gb subunit that acts as a positive Epistasis testing: Epistasis tests were conducted by examining progeny from tetrad dissections of crosses of RWP4 regulator of the gpa2 Ga (Landry et al. 2000). (gpa2 ) with strains carrying mutations in git1, git2, git3, In this article, we further characterize the genetic ingit5, pka1/git6, git7, and git10. Following germination and colteractions between gpa2 and the other upstream git ony formation, progeny were transferred to a fresh YEA plate, genes through the use of an “activated” allele of gpa2 grown 1 day, and then replica plated to 5-FOA-containing whose product is defective in its autoinhibitory GTPase medium. 5-FOA resistance was determined 2–3 days after replica plating. activity. Furthermore, we describe the cloning and char-
G 蛋白 α 和 β γ 亚基对哺乳动物腺苷酸环化酶的调节。
DOI：
10.1101/sqb.1992.057.01.017
发表时间：
1992
期刊：
Cold Spring Harbor symposia on quantitative biology
影响因子：
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作者：
Tang,WJ;Iñiguez-Lluhi,JA;Mumby,S;Gilman,AG
通讯作者：
Gilman,AG