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

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 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 ( to study cyclic nucleotide-associated signal transduction cascades).

Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta

“…These results are also reported here. Yoshida et al 34 also report a small amount of light-dependent phosphodiesterase activity under in vitro conditions with membranes isolated from transfected HEK293 cells. While we were unable to detect light dependence, even under conditions identical to those of Yoshida et al (Figure 6), we do not view this as a significant difference between the two studies.…”

“…Very recently, Yoshida et al 34 published a study of this same protein from S. rosetta , the only other paper published on this protein. There are several similarities between their study and ours.…”

“…There are several similarities between their study and ours. Yoshida et al 34 report heterologous expression of the protein in HEK293 cells and purification by immunoaffinity chromatography using an epitope tag and the 1D4 antibody; the absorption spectrum after addition of ATR, with a maximum at 490 nm and a shoulder at 460 nm; the phosphodiesterase activity that is selective for cGMP over cAMP; and, finally, the phosphodiesterase activity, for both cGMP and cAMP, that is constitutive and present without exposure to light. These results are also reported here.…”

Signal transduction in photoreceptor histidine kinases

“…Whereas SRs are indirectly coupled to histidine kinases via the Htr transducers, several rhodopsins are covalently linked to histidine kinase effectors, for example HKR1 from Chlamydomonas reinhardtii. 120 CrHKR1 and related rhodopsins with covalently attached nucleotide cyclase 121 and phosphodiesterase effectors 118,122 are subsumed as enzymerhodopsins. 123 As functional data on enzymerhodopsins are sparse and no structural information is available yet, the focus of the present discussion will be on SR.…”

Light-regulated collective contractility in a multicellular choanoflagellate

“…Although choanoflagellates are unpigmented and transparent, at least four choanoflagellates (9) encode a choanoflagellate-specific rhodopsin-phosphodiesterase fusion protein ( Fig. S2), RhoPDE, that has been investigated for its potential as an optogenetic tool (20)(21)(22)(23). RhoPDE proteins consist of an N-terminal type I (bacterial) rhodopsin (a class of photosensitive transmembrane proteins broadly involved in light detection (24)) fused to a Cterminal phosphodiesterase (PDE) that catalyzes light-dependent cyclic nucleotide hydrolysis (25), which act on the timescale of transcriptional regulation (at least several minutes (26,27)) and therefore appear unlikely to mediate the light-to-dark transition response.…”