Keiji Seno

Presence of Rhodopsin and Porphyropsin in the Eyes of 164 Fishes, Representing Marine, Diadromous, Coastal and Freshwater Species—A Qualitative and Comparative Study

There are two types of visual pigments in fish eyes; most marine fishes have rhodopsin, while most freshwater fishes have porphyropsin. The biochemical basis for this dichotomy is the nature of the chromophores, retinal (A1) and 3‐dehydroretinal (A2), each of which is bound by an opsin. In order to study the regional distribution of these visual pigments, we performed a new survey of the visual pigment chromophores in the eyes of many species of fish. Fish eyes from 164 species were used to examine their chromophores by high‐performance liquid chromatography—44 species of freshwater fish, 20 of peripheral freshwater fish (coastal species), 10 of diadromous fish and 90 of seawater fish (marine species) were studied. The eyes of freshwater fish, limb freshwater fish and diadromous fish had both A1 and A2 chromophores, whereas those of marine fish possessed only A1 chromophores. Our results are similar to those of previous studies; however, we made a new finding that fish which live in freshwater possessed A1 if living near the sea and A2 if living far from the sea if they possessed only one type of chromophore.

Changes in Lipid Droplet Localization During Embryogenesis of the Silkworm, Bombyx mori

Abstract Lipid droplets are considered one of the most important energy sources in lepidopteran eggs during late embryogenesis, but the process of their incorporation into the embryo is as yet unknown. The present study focused on the process of transition of lipid droplets from the extraembryonic yolk to the embryo of the silkworm Bombyx mori, using morphological and biochemical approaches. The morphological study revealed that the incorporation of lipid droplets from the extraembryonic yolk into the embryo occurs at three points and in three different ways during the development of the embryo. Some lipid droplets were translocated directly from the extraembryonic yolk to the embryo before the blastokinesis stage. However, the majority of lipid droplets together with the other components of the extraembryonic yolk were incorporated in the embryo via both morphogenetic inclusion during dorsal closure and ingestion of the extraembyonic yolk by the developing caterpillar prior to hatching. Similar results were obtained from the biochemical study. Thus, we propose that there are three steps in the incorporation of lipid droplets from the extraembryonic yolk into the embryo. In addition, morphological and biochemical data concerning the total amount of lipid droplets in the egg suggested that lipid droplets were mainly consumed during late embryogenesis, seeming to synchronize with tracheal development.

Palmitoylation is a prerequisite for dimerization-dependent raftophilicity of rhodopsin

The visual photopigment rhodopsin (Rh) is a prototypical G protein–coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (i.e. raftophilicity) upon light-dependent binding with the cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and that Rh is posttranslationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, using biochemical assays, we found that Rh*–Gt complexes in the detergent-resistant membrane are partially resistant to cholesterol depletion by methyl-β-cyclodextrin and that the Rh-to-Gt stoichiometry in this methyl-β-cyclodextrin–resistant complex is 2:1. Next, we found that IgG-mediated Rh–Rh cross-linking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Rh depalmitoylation via reduction of thioester linkages blocked the translocation of IgG–cross-linked Rh to the detergent-resistant membrane, highlighting that the two palmitoyl moieties are important for the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs such as Rh can acquire raftophilicity upon G protein–stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.

Methyl-β-cyclodextrin is a useful compound for extraction and purification of prenylated enzymes from the retinal disc membrane

cGMP phosphodiesterase 6 (PDE6) and rhodopsin kinase (GRK1) are quantitatively minor prenylated proteins involved in vertebrate phototransduction. Here, we report that methyl-β-cyclodextrin (MCD), a torus-shaped oligosaccharide with a hydrophobic pore, can be used as a selective extractant for such prenylated proteins from frog retinal disc membranes, and that MCD makes it possible to purify frog PDE6 holoenzyme with very simple procedure. The EC50s of MCD for the extraction of GRK1 and PDE6 from the cytoplasmic surface of the disc membrane were 0.17 and 5.1 mM, respectively. By successive extraction of the membrane by 1 mM and then 20 mM MCD, we obtained crude GRK1 and PDE6, respectively. From the 20mM extract, we were able to purify the PDE6 holoenzyme using one-step anion-exchange column chromatography. From 1mM MCD extract, GRK1 was further purified by an affinity column. Following the removal of MCD by ultrafiltration, we were able to confirm integrity of these enzymes by reconstituting phototransduction system in vitro. We have therefore demonstrated that MCD is a useful compound for selective extraction and purification of prenylated peripheral membrane proteins from the cytoplasmic surface of biological membranes.