Refine
Has Fulltext
- yes (2)
Is part of the Bibliography
- yes (2)
Document Type
- Journal article (2)
Language
- English (2)
Keywords
- membrane proteins (2) (remove)
Institute
An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca\(^{2+}\)-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca\(^{2+}\) by tandem endosomes into the cytosol via CatCh was visualized using the Ca\(^{2+}\)-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca\(^{2+}\) in response to light.
Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO\(^{-}\) mutant and carO\(^{+}\) control strains showed a faster development of light-exposed carO-germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.