We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradiation with UV or violet light (350 -400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest.

The inositol 1,4,5-trisphosphate receptor (IP3R) exists as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca2+ channel. Molecular cloning studies have shown that there are at least three types of IP3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP3R subunit. We found that all the three types of IP3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP3Rs form heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP3R subunits assemble to form heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP3R types, suggesting the possibility that various compositions of subunits show distinct channel properties. The diversity of IP3R channels may be further increased by the co-assembly of different IP3R subunits to form homo- or heterotetramers.

Wingless (Wg)/Wnt has been proposed to exert various functions as a morphogen depending on the levels of its signalling. Therefore, not just the concentration of Wg/Wnt, but also the responsiveness of Wg/Wnt-target cells to the ligand, must have a crucial function in controlling cellular outputs. Here, we show that a balance of ubiquitylation and deubiquitylation of the Wg/Wnt receptor Frizzled determines the cellular responsiveness to Wg/Wnt both in mammalian cells and in Drosophila, and that the cell surface level of Frizzled is regulated by deubiquitylating enzyme UBPY/ubiquitin-specific protease 8 (USP8). Although ubiquitylated Frizzled underwent lysosomal trafficking and degradation, UBPY/USP8-dependent deubiquitylation led to recycling of Frizzled to the plasma membrane, thereby elevating its surface level. Importantly, a gain and loss of UBPY/USP8 function led to up- and down-regulation, respectively, of canonical Wg/Wnt signalling. These results unveil a novel mechanism that regulates the cellular responsiveness to Wg/Wnt by controlling the cell surface level of Frizzled.

We determined the amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 Ins(1,4,5)P3 receptor (IP3R1). We expressed various parts of IP3R1 from deleted cDNA and examined their CaM-binding ability. It was shown that the sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length IP3R1 with replacement of Trp-1576 by Ala lost its CaM-binding ability. Antibody against residues 1564-1585 of IP3R1 inhibited cerebellar IP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifted when Ca(2+)-CaM was added. From the change in the fluorescence spectrum, we estimated the dissociation constant (KD) between the peptide and CaM to be 0.7 microM. The submicromolar value of KD suggests an actual interaction between CaM and IP3R1 within cells. The CaM-binding ability of other types of IP3Rs was also examined. A part of the type 2IP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also bound CaM, while the expressed full-length type 3 IP3R did not.

We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "AzamiGreen (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric greenemitting fluorescent protein comparable to EGFP.

A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. Here we show that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disk cells are functionally diverse. The UDP-sugar transporter FRINGE-CONNECTION (FRC) is localized to a subset of the Golgi units distinct from those harboring SULFATELESS (SFL), which modifies glucosaminoglycans (GAGs), and from those harboring the protease RHOMBOID (RHO), which processes the glycoprotein SPITZ (SPI). Whereas the glycosylation and function of NOTCH are affected in imaginal disks of frc mutants, those of SPI and of GAG core proteins are not, even though FRC transports a broad range of glycosylation substrates, suggesting that Golgi units containing FRC and those containing SFL or RHO are functionally separable. Distinct Golgi units containing FRC and RHO in embryos could also be separated biochemically by immunoisolation techniques. We also show that Tn-antigen glycan is localized only in a subset of the Golgi units distributed basally in a polarized cell. We propose that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification.fringe connection ͉ glycosylation ͉ posttranslational modification T he pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are Ͼ300 glycosylenzymes in humans and Ͼ100 in Drosophila, but is their enzymatic specificity sufficient to explain the precise modification of all substrates? One possible mechanism that might also contribute to the specific (and diverse) pattern of glycosylation would be the localization͞compartmentalization of glycosylenzymes.The Golgi complex, where protein glycosylation takes place, has been regarded as a single functional unit, consisting of cis-, medial-, and transcisternae in mammalian cells. However, the three-dimensional reconstruction of electron microscopic images of the mammalian Golgi structure has suggested the existence of more than one Golgi stack, with the individual stacks being connected into a ribbon by tubules bridging equivalent cisternae (1). Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly (2, 3). In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase (4, 5), although there has been no evidence to date to indicate functional differences among the Golgi fragments.We previously reported a Drosophila UDP-sugar transporter, FRINGE CONNECTION (FRC), that transports a broad range of UDP-sugars that can be used for the synthesis of various glycans, including N-linked types, GAGs, and mucin types (6, 7). Interestingly, despite its broad specificity, loss-of-function studies have revealed that FRC is selectively required for Notch glycosylation, but not for GA...

Monoclonal antibodies were raised that specifically recognize the COOH-terminal sequences and the loop sequences between the fifth and the sixth transmembrane spanning regions of human inositol1,4,5-trisphosphate receptor (IP,R) type 1,2 and 3. Western blot analysis using Jurkat cells, mouse cerebellum, COS-7 expressing IP,R type 3 cDNA showed that those monoclonal antibodies reacted specifically with each of these three IP,R subtypes and that they do not cross-react. These antibodies could be used for the specific immunoprecipitation of IP,Rs. Using these monoclonal antibodies, the expression profiles of IP,R-subtype proteins were found to be different among inflammatory cells such as macrophages, polymorphonuclear cells, mast cells, eosinophils, splenocytes, thymocytes and megakaryocytic cells. Usually, more than one type of IP,R were expressed in a cell simultaneously. The observation of CMK cells under immunofluorescence confocal microscopy revealed that IP,R type 1 and type 2 are located at different subcellular fractions.

Inositol I ,4,Qrisphosphate (IP,)-mediated Ca2+ signaling plays important roles in cellular responses to extracellular stimuli. We recently succeeded in cloning human counterparts of the three subtypes derived from separate genes. Using the cDNA sequences type-specific to these subtype receptors, we here analyzed the expression profile of IP,R subtypes in stimulated and unstimulated human hematopoietic cell lines representing T cells, B cells, neutrophils, macrophages, erythrocytes and megakaryocytes. Northern and dot blot analysis showed that each IP,R subtype is expressed differently in these cells and that the expression profile in each cell is dynamically changed upon stimuli which induce differentiation. Moreover, most of these cells were found to simultaneously express at least two different subtype receptors.