Analyses of Structure of Photoreceptor Organelle and Blepharismin-associated Protein in Unicellular Eukaryote Blepharisma¶

Abstract

In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as "blepharismins" (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules' three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyblepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment-200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Blepharisma.

Primary Photoprocesses Involved in the Sensory Protein for the Photophobic Response ofBlepharisma japonicum

“…Blue cells were washed, collected by low speed centrifugation, and resuspended in a 20 mmol L −1 sodium cholate (NaCh) solution. The OBIP chromoprotein was purified by liquid chromatography of this preparation on a hydroxyapatite column. ,, The protocol closely follows the one described by Checcucci et al, which proved to yield samples mainly containing the 200 kDa (SDS-PAGE determination) OBIP protein. The purity of our sample was not quantified.…”

“…Studying the primary photoprocesses involved in biological photoreceptors is a fascinating field that teaches us on the various solutions evolved by Nature in order to convert light into a biological signal. Most of the known light-sensing proteins belong to one of the following six families: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF proteins. , The chromoprotein thought to be responsible for the step-up photophobic response of Blepharisma japonicum − nevertheless belongs to a different family. B. japonicum is a red-colored photophobic ciliated protozoan, the biology of which was described by Giese…”

“…B. japonicum is a red-colored photophobic ciliated protozoan, the biology of which was described by Giese ,, Blue cells are particularly interesting because they still display the same phobic response as red cells and because a stable chromophore−protein complex could be extracted from them. , This complex is made of a nonsoluble 200 kDa protein, noncovalently bound to the chromophore identified as responsible for the photophobic response, namely oxyblepharismin (OxyBP, see chemical structure in Scheme ). , OxyBP is a phenanthroperylene quinone derivative close to hypericin (Hyp, Scheme ), a well-known photosensitizing agent . OxyBP is also the photooxidized form of blepharismin (BP, Scheme ), which is the photoreceptor molecule of the dark-adapted (red-colored) form of B. japonicum . , We will refer to the OxyBP-protein complex of B. japonicum as OBIP (OxyBlepharismin bInding Protein).…”

“…Most of the known light-sensing proteins belong to one of the following six families: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF proteins. 1,2 The chromoprotein thought to be responsible for the step-up photophobic response of Blepharisma japonicum [3][4][5][6][7] nevertheless belongs to a different family. B. japonicum is a red-colored photophobic ciliated protozoan, the biology of which was described by Giese.…”

Hypericin‐Like Photoreceptors

“…Fabczak et al (2001) reported that phosphorylation of two other proteins, 28and 46-kDa proteins, of B. japonicum is down-and up-regulated by light, respectively. They suggest that the phosphorylated; 46-kDa protein may associate with or be a proteolytic digest product from the 200-kDa protein (Matsuoka et al, 2000). The large discrepancy between these two values of mol wt has not been resolved.…”