Collaborative Research: Signaling Via Opsins and Opsin-Related Proteins in Fungi

合作研究：通过真菌中的视蛋白和视蛋白相关蛋白进行信号传导

• 批准号: 0078282
• 负责人: Katherine Borkovich
• 金额: $ 42万
• 依托单位: The University of Texas Health Science Center at Houston
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0078282&HistoricalAwards=false
• 关键词: Collaborative; Research; Signaling; Via; Opsins

Opsins are seven-transmembrane helix proteins that bind all-trans or 11-cis isomers of retinal to form light-absorbing pigments known as rhodopsins. Previously, opsin-encoding genes had only been cloned from animals and the archaea. The principal investigator recently identified the first opsin gene from eukaryotic microbes, Neurospora crassa nop-1. The NOP-1 protein sequence is most similar to that of archaeal opsins, with conservation of all 22 retinal binding pocket residues, including the lysine residue that forms a Schiff base linkage with retinal. NOP-1 is also similar to several predicted proteins from various fungal species, including Saccharomyces cerevisiae Hsp30p. With the exception of two predicted proteins from filamentous fungi, all other related fungal proteins lack the Schiff base lysine residue and we have referred to them as Opsin-Related Proteins (ORPs). NOP-1 has been overexpressed in Pichia pastoris and its spectral properties determined. Similar to archaeal opsins, NOP-1 binds all-trans retinal with a Schiff base linkage. The resultant pigment has an absorption maximum at 534 nm (green). The relatively long photocycle of the NOP-1 pigment is similar to that of archaeal sensory rhodopsins, suggesting NOP-1 functions as a sensory receptor in N. crassa. The nop-1 message is most abundant under conditions that favor asexual sporulation (conidiation) in N. crassa. Although ?nop-1 mutants do not have visible phenotypes at 30oC, these strains exhibit green light-dependent defects in asexual spore-forming structures and cell viability at elevated growth temperatures (37-42oC). Also, the principal investigator identified an EST encoding a N. crassa ORP (orp-1). The predicted ORP-1 protein sequence is most similar to HSP30 from Coriolus versicolor. In contrast to NOP-1, ORP-1 only shows conservation of 50% of the 22 retinal binding pocket residues with archaeal rhodopsins, including substitution of isoleucine for the Schiff base lysine residue. The sequence similarity between NOP-1 and archaeal rhodopsins; the evolutionary relationship between NOP-1 and S. cerevisiae Hsp30p; the light-dependent expression of nop-1; the sensory rhodopsin-like photocycle of NOP-1; the light-dependent conidiation defect of ?nop-1 mutants in the presence of oligomycin, and the light-dependent defects in cell growth at elevated temperatures lead to the following hypothesis: Opsins and ORPs regulate cell growth, viability and development during cellular stress responses in N. crassa, potentially via light-dependent and independent pathways, respectively. The Research Objectives of this project are 1) To determine the localization and native chromophore of NOP-1 in N. crassa and to further characterize the involvement of nop-1 in stress and developmental regulation; 2) to identify genes that regulate nop-1 gene expression and genes whose expression is regulated by nop-1; 3) to create and analyze a ?orp-1 mutant and to over-express and characterize the encoded ORP-1 protein, and 4) to identify other genes encoding opsins and ORPs, and other nop-1 pathway components. During this collaborative research project, one laboratory will focus on Objectives 1 and 3, while the other will focus on Objectives 2 and 4. This collaborative research project will strengthen and extend the existing collaboration, and exploit the complementary skills and resources available to the two principal investigators. One principal investigator has extensive expertise in genome analysis, molecular evolution, the genetics and biochemistry of stress responses and the genetics of filamentous fungi in general. The other has significant experience in protein biochemistry, mutational analysis of fungal genes, stress responses and signal transduction via both G proteins and histidine kinases. The former has access to the resources of the Neurospora Genome Project, including automated sequencing and microarray facilities. The latter is in close proximity to a leader in biophysical analysis of archaeal rhodopsins and who has been a past and is a current collaborator. The existence of seven-helix opsin receptors has been postulated in eukaryotic microbes for decades. Many of these organisms utilize light signals to regulate cell growth, reproduction, phototaxis or the circadian clock. Therefore, the identification of an opsin in N. crassa has enormous repercussions for filamentous fungi and other eukaryotes. Furthermore, the lack of an opsin in the yeast Saccharomyces cerevisiae points to the importance of studying this class of proteins in the model filamentous fungus N. crassa. In addition, very little is known about ORPs in fungi: for example, whether they can bind a chromophore, absorb light, etc. Thus, the principal investigators are in the position of making pivotal discoveries in this field using N. crassa as an experimental organism.

视蛋白是七跨膜螺旋蛋白，其结合视网膜的全反式或11-顺式异构体以形成称为视紫红质的光吸收色素。 以前，编码视蛋白的基因只从动物和古细菌中克隆出来。 首席研究员最近从真核微生物中鉴定了第一个视蛋白基因，粗糙脉孢菌nop-1。 NOP-1蛋白质序列与古细菌视蛋白的序列最相似，保留了所有22个视网膜结合口袋残基，包括与视网膜形成席夫碱连接的赖氨酸残基。 NOP-1也类似于来自各种真菌物种的几种预测蛋白，包括酿酒酵母Hsp 30 p。 除了来自丝状真菌的两种预测的蛋白质之外，所有其他相关的真菌蛋白质都缺乏席夫碱赖氨酸残基，并且我们将它们称为视蛋白相关蛋白质（ORP）。NOP-1已在毕赤酵母中过表达并测定其光谱性质。 与古细菌视蛋白类似，NOP-1与具有席夫碱键的全反式视黄醇结合。 所得颜料在534 nm（绿色）处具有最大吸收。 NOP-1色素的相对较长的光周期与古细菌的感觉视紫红质相似，表明NOP-1在N.粗鲁。nop-1信息在有利于N.粗鲁。 虽然？nop-1突变体在30 ℃下没有可见的表型，这些菌株在升高的生长温度（37- 42 ℃）下在无性孢子形成结构和细胞活力方面表现出绿色光依赖性缺陷。此外，主要研究者确定了编码N的EST。crassa ORP（orp-1）。 预测的ORP-1蛋白质序列与来自云芝的HSP 30最相似。 与NOP-1相反，ORP-1仅显示出与古细菌视紫红质的22个视网膜结合口袋残基中的50%的保守性，包括异亮氨酸取代席夫碱赖氨酸残基。NOP-1与古细菌视紫红质的序列相似性，NOP-1与S.酿酒酵母Hsp 30 p; nop-1的光依赖性表达、nop-1的感觉性视紫红质样光循环、NOP-1的光依赖性分生孢子缺陷、NOP-1的光依赖性表达、nop-1的感觉性视紫红质样光循环、nop-1的光依赖性分生孢子缺陷等。nop-1在寡霉素存在下的突变，以及在高温下细胞生长的光依赖性缺陷导致以下假设：视蛋白和ORP调节N. crassa，可能通过光依赖性和独立的途径，分别。 本课题的研究目的是：1）确定NOP-1在烟草中的定位和天然发色团; crassa和进一步表征参与的nop-1的压力和发育调控; 2）确定基因的调节nop-1基因的表达和基因的表达受到nop-1的调控; 3）创建和分析一个？orp-1突变体，并过表达和表征编码的ORP-1蛋白，和4）鉴定编码视蛋白和ORP的其它基因，以及其它nop-1途径组分。 在这个合作研究项目中，一个实验室将专注于目标1和3，而另一个实验室将专注于目标2和4。 这一合作研究项目将加强和扩大现有的合作，并利用两名主要调查员的互补技能和资源。 其中一名首席研究员在基因组分析、分子进化、应激反应的遗传学和生物化学以及丝状真菌的遗传学方面具有广泛的专业知识。 另一位在蛋白质生物化学、真菌基因突变分析、胁迫反应和通过G蛋白和组氨酸激酶的信号转导方面具有重要经验。 前者可以使用脉孢菌基因组计划的资源，包括自动测序和微阵列设施。 后者与古细菌视紫红质生物物理分析的领导者非常接近，他过去是现在的合作者。在真核微生物中存在七螺旋视蛋白受体已经被假设了几十年。 这些生物体中的许多利用光信号来调节细胞生长、繁殖、趋光性或生物钟。 因此，在N. crassa对丝状真菌和其他真核生物有巨大的影响。 此外，在酵母酿酒酵母中缺乏视蛋白，这表明在模式丝状真菌N.粗鲁。 此外，对真菌中的ORP知之甚少：例如，它们是否可以结合发色团，吸收光等。crassa作为一种实验生物。