A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity

Kazuho Yoshida,Satoshi P Tsunoda,Leonid S Brown,Hideki Kandori

doi:10.1074/jbc.m117.775569

Abstract

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising second-generation tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).

Highlights

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising secondgeneration tools in optogenetics
We propose that rhodopsin fused to a C-terminal PDE domain (Rh-PDE) is a microbial rhodopsin that binds all-trans-retinal as a chromophore, where light absorption triggers protein structural changes to induce the enzymatic activity
We report on a novel type of microbial rhodopsin from the choanoflagellate S. rosetta containing a C-terminal PDE domain

Summary

Results

The genome of the choanoflagellate S. rosetta (sequenced by the Broad Institute, NCBI accession number PRJNA37927) contains an unusual gene (NCBI Gene ID 16078606) encoding a rhodopsin fused to a C-terminal PDE domain (Rh-PDE). Upon application of the 510-nm light, the cells expressing mosquito Opn show a strong decrease in the intracellular cAMP concentration, which continues to decrease after the light is switched off, reaching a minimum in ϳ5 min, and recovers with ␶1/e of 16 min (Fig. 3A, green line) These data reproduce the data reported previously [27], and it is obvious that the luminescence signal is smaller and faster (both in the rise and the decay) in the case of Rh-PDE under the same illumination conditions (Fig. 3A, red line). The arrow corresponds to about 70 kDa. B, absorption spectrum of purified full-length Rh-PDE (dotted line; reproduced from the black line in A) superimposed with the action spectrum of the light-induced enzymatic activity toward cAMP in HEK293T cells (the peak amplitudes in Fig. 3D are plotted versus irradiation wavelength with 0.4 ␮W mmϪ2 intensity). This result indicates that Rh-PDE forms stable oligomers that survive solubilization

Discussion

Molecular biology

Estimation of chromophore regeneration

Phylogenetic analysis of rhodopsin genes