The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae.

Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation, as an additional gene involved in prospore membrane extension. Genetic and cell biological analyses suggested that Spo73 functions on the prospore membrane with other factors in prospore membrane extension, counteracting the bending force of the prospore membrane. Spo73 is the first dysferlin domain-only protein ever analyzed. The dysferlin domain is conserved from yeast to mammals and is found in dysferlin proteins, which are involved in dysferlinopathy, although the precise function of the domain is unknown. Continued analysis of Spo73 will contribute to our understanding of the function of dysferlin domains and dysferlinopathy.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

Comparative study on the nuclear hormone receptor activity of various phytochemicals and their metabolites by reporter gene assays using Chinese hamster ovary cells.

Phytochemicals are naturally present in a wide variety of plants, and have been suggested to exert a number of effects beneficial to human health. Several phytochemicals possess estrogenic activity through estrogen receptor alpha (ERalpha) and ERbeta, and are, therefore, termed phytoestrogens. In this study, we examined whether various phytochemicals have agonistic and/or antagonistic activity against six human nuclear receptors (ERalpha, ERbeta, androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor alpha(1) (TRalpha(1)) and TRbeta(1)) by in vitro reporter gene assays using Chinese hamster ovary cells. Of the 31 phytochemicals tested, including flavonoids, isoflavonoids, coumestan, lignans, catechins and their metabolites, 20 compounds showed estrogenic activity via ERalpha and/or ERbeta, and we ranked these phytochemicals according to their estrogenic potency via ERalpha and ERbeta. As a result, coumestrol and genistein strongly activated ERalpha and ERbeta at very low concentrations of <1x10(-10) M. Most phytochemicals showing estrogenic activity also exhibited agonistic activity against ERbeta at lower concentrations than those for ERalpha, and two typical isoflavones, genistein and daidzein, in particular, showed a potent preference for ERbeta. Further, we found that baicalein has ERbeta antagonistic activity, and two compounds, enterolacton and O-desmethylangolensin, have AR antagonistic activity. Nevertheless, none of tested compounds showed AR agonistic activity together with GR, TRalpha(1) and TRbeta(1) agonistic/antagonistic activity. These results suggest that various phytochemicals or their metabolites preferentially interact with ERalpha/beta among the six nuclear hormone receptors tested, and that the ERbeta agonistic activity, in particular, of these compounds may be associated with various beneficial effects on human health.

In vitro screening of 200 pesticides for agonistic activity via mouse peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma and quantitative analysis of in vivo induction pathway.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors and key regulators of lipid metabolism and cell differentiation. However, there have been few studies reporting on a variety of environmental chemicals, which may interact with these receptors. In the present study, we characterized mouse PPARalpha and PPARgamma agonistic activities of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 11 acid amides, 7 triazines, 8 ureas and 44 others) by in vitro reporter gene assays using CV-1 monkey kidney cells. Three of the 200 pesticides, diclofop-methyl, pyrethrins and imazalil, which have different chemical structures, showed PPARalpha-mediated transcriptional activities in a dose-dependent manner. On the other hand, none of the 200 pesticides showed PPARgamma agonistic activity at concentrations

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization.

Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses. Secondary structure prediction showed that the second putative transmembrane helices (helix B) of these rhodopsins are rich in glutamate residues, and homology modeling suggested that some additional intra- or intermolecular interactions are necessary for opsin-like folding of the N-terminal ca. 300-aa membrane spanning domains of 712 and 737-aa polypeptides. These results complement physiological and electrophysiological experiments combined with the manipulation of their expression [O.A. Sineshchekov, K.H. Jung, J.H. Spudich, Proc. Natl. Sci. USA 99 (2002) 8689; G. Nagel, D. Olig, M. Fuhrmann, S. Kateriya, A.M. Musti, E. Bamberg, P. Hegemann, Science 296 (2002) 2395].