Busse Wayne, Klotzsch Enrico, Kamrani Yousef Yari, Wordtmann Natalie, Kelterborn Simon, Hegemann Peter, Broser Matthias
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115, Berlin, Germany.
Institute of Biology, Mechanobiology, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115, Berlin, Germany.
EMBO J. 2025 May 27. doi: 10.1038/s44318-025-00452-x.
Photoorientation in motile fungal zoospores is mediated by rhodopsin guanylyl cyclases (RGCs). In certain chytrids, these photoreceptors form heterodimers consisting of a visible-light-absorbing RGC paired with neorhodopsin (NeoR), a rhodopsin distinguished by its unique spectral properties: far-red absorption and high fluorescence. Leveraging the native fluorescence of NeoR, we detected RGCs in living zoospores of the fungus Rhizoclosmatium globosum. The reversible photoswitching of bistable NeoR enabled super-resolution microscopy, facilitating single-molecule detection and quantification of NeoR proteins within individual zoospores. This approach also revealed the precise localization of RGCs within the rumposome, a chytrid-specific organelle hypothesized to mediate photoreception. Fluorescence tracking across different stages of the chytrid life cycle and the analysis of transcriptomic data confirmed that RGCs are predominantly present during the zoospore stage. Functional assays of recombinantly expressed RGC heterodimers with modified substrate specificity revealed that only one of the two pseudo-symmetric nucleotide-binding sites is catalytically active. Strikingly, disrupting nucleotide binding in the non-catalytic site enhanced light-triggered cyclase activity by up to ninefold, indicating an allosteric regulatory mechanism in heterodimeric RGCs.
游动真菌游动孢子中的光取向由视紫红质鸟苷酸环化酶（RGCs）介导。在某些壶菌中，这些光感受器形成异源二聚体，由一个吸收可见光的RGC与新视紫红质（NeoR）配对组成，新视紫红质是一种视紫红质，其独特的光谱特性使其与众不同：远红光吸收和高荧光。利用NeoR的天然荧光，我们在球形根肿菌的活游动孢子中检测到了RGCs。双稳态NeoR的可逆光开关实现了超分辨率显微镜，有助于在单个游动孢子内对NeoR蛋白进行单分子检测和定量。这种方法还揭示了RGCs在rumposome内的精确定位，rumposome是一种壶菌特有的细胞器，据推测可介导光感受。对壶菌生命周期不同阶段的荧光追踪以及转录组数据分析证实，RGCs主要存在于游动孢子阶段。对具有修饰底物特异性的重组表达RGC异源二聚体的功能测定表明，两个假对称核苷酸结合位点中只有一个具有催化活性。令人惊讶的是，破坏非催化位点的核苷酸结合可使光触发的环化酶活性提高多达九倍，这表明异源二聚体RGCs中存在变构调节机制。
