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

视蛋白是7个跨膜螺旋蛋白，与视网膜的全反式或11顺式异构体结合，形成被称为视紫红质的吸光色素。此前，视蛋白编码基因仅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神经孢子虫（Neurospora crassa nop-1）中发现了第一个视蛋白基因。NOP-1蛋白序列与古菌视蛋白最为相似，保留了所有22个视网膜结合袋残基，包括与视网膜形成希夫碱连锁的赖氨酸残基。NOP-1也与来自各种真菌物种的几种预测蛋白相似，包括酿酒酵母Hsp30p。除了两种丝状真菌的预测蛋白外，其他所有相关的真菌蛋白都缺乏希夫碱赖氨酸残基，我们将它们称为视蛋白相关蛋白（Opsin-Related proteins, orp）。NOP-1在毕赤酵母中过表达，并测定了其光谱特性。与古细菌视蛋白类似，NOP-1通过一个希夫碱基键结合全反式视网膜。合成的颜料在534 nm（绿色）处具有最大吸收。NOP-1色素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在草属植物中具有感觉受体的功能。n -1信息在有利于无性孢子形成（分生）的条件下最为丰富。虽然?nop-1突变体在30℃下没有明显的表型，这些菌株在高温（37-42℃）下无性孢子形成结构和细胞活力表现出依赖绿光的缺陷。此外，研究人员还发现了一个编码N. crassa ORP （ORP -1）的EST。预测的ORP-1蛋白序列与花斑Coriolus versicolor的HSP30最相似。与NOP-1相比，ORP-1只有50%的22个视网膜结合袋残基与古菌紫红质保持一致，包括异亮氨酸取代希夫碱赖氨酸残基。NOP-1与古细菌视紫红质序列的相似性NOP-1与酿酒链球菌Hsp30p的进化关系；nop-1的光依赖性表达；NOP-1的感觉视紫红质样光循环；的光依赖条件缺陷？寡霉素存在下的nop-1突变体，以及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分别通过光依赖性和非依赖性途径调节细胞在细胞应激反应中的生长、活力和发育。本课题的研究目标是：1)确定NOP-1在草属植物中的定位和天然发色团，进一步表征NOP-1在胁迫和发育调控中的作用；2)鉴定调控nop-1基因表达的基因和受nop-1基因表达调控的基因；3)创建和分析一个？orp-1突变体，并对编码的orp-1蛋白进行过表达和表征；4)鉴定其他编码视蛋白和orp的基因，以及其他nop-1通路组分。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。这个合作研究项目将加强和扩展现有的合作，并利用两位主要研究人员的互补技能和资源。一位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G蛋白和组氨酸激酶信号转导方面具有丰富的经验。前者可以使用神经孢子菌基因组计划的资源，包括自动测序和微阵列设备。后者在古细菌视紫红质生物物理分析方面的领导地位非常接近，他过去和现在都是合作者。真核微生物中存在七螺旋视蛋白受体的假设已经存在了几十年。许多生物利用光信号来调节细胞生长、繁殖、趋光性或生物钟。因此，鉴定一种视蛋白对丝状真菌和其他真核生物具有巨大的影响。此外，在酿酒酵母中缺乏视蛋白表明了在模式丝状真菌N. crassa中研究这类蛋白质的重要性。此外，对于真菌中的orp，我们所知甚少：例如，它们能否结合发色团，能否吸收光等。因此，主要研究人员在这个领域的关键发现的位置上使用N. crassa作为实验生物。

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.