Jing Yu-Strzelczyk

Opsins are known as proteins that bind retinal to form light-sensing rhodopsins, which mediate diverse forms of photoreception and are widely used for optogenetic control of cellular activity. Yet many opsins lack canonical retinal-binding features or, in their native biological contexts, are expressed where chromophore availability is limited, raising the question whether retinal-free opsins are silent. Here we show that retinal-free ion-transporting microbial opsins are not inert but adopt intrinsic, light-independent functional states alongside their canonical light-driven activity. Using developmental phenotyping in transgenic Drosophila melanogaster, electrophysiology in Xenopus oocytes and mammalian cells, together with structural and computational analyses, we demonstrate robust light-independent ion conductance in multiple channel-type opsins and an ion-pumping opsin. Structural modeling and molecular dynamics simulations reveal conformational rearrangements in transmembrane helices and internal water networks that are consistent with stabilization of apo-conductive states. Together, these findings establish apo-opsin activity as a defined functional state of microbial opsins, expand the current framework of rhodopsin biology, and highlight the need to consider chromophore-independent activity in both basic research and optogenetic applications.

Early plant responses to different stress situations often encompass cytosolic Ca²⁺ increases, plasma membrane depolarization and the generation of reactive oxygen species^1–3. However, the mechanisms by which these signalling elements are translated into defined physiological outcomes are poorly understood. Here, to study the basis for encoding of specificity in plant signal processing, we used light-gated ion channels (channelrhodopsins). We developed a genetically engineered channelrhodopsin variant called XXM 2.0 with high Ca²⁺ conductance that enabled triggering cytosolic Ca²⁺ elevations in planta. Plant responses to light-induced Ca²⁺ influx through XXM 2.0 were studied side by side with effects caused by an anion efflux through the light-gated anion channelrhodopsin ACR1 2.0⁴. Although both tools triggered membrane depolarizations, their activation led to distinct plant stress responses: XXM 2.0-induced Ca²⁺ signals stimulated production of reactive oxygen species and defence mechanisms; ACR1 2.0-mediated anion efflux triggered drought stress responses. Our findings imply that discrete Ca²⁺ signals and anion efflux serve as triggers for specific metabolic and transcriptional reprogramming enabling plants to adapt to particular stress situations. Our optogenetics approach unveiled that within plant leaves, distinct physiological responses are triggered by specific ion fluxes, which are accompanied by similar electrical signals.

Plasmodium falciparum -infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes not only facilitate movement of nutrients, metabolites, and other molecules between these compartments, but also are common therapeutic targets and can confer antimalarial drug resistance.

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum , an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures – the food vacuole, the apicoplast, and the parasite plasma membrane – and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium -specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium . Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions. Importance: Plasmodium falciparum -infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes do not only facilitate movement of nutrients, metabolites and other molecules between these compartments, but are common therapeutic targets and can also confer antimalarial drug resistance. Orphan membrane transporter in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent to host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed into their importance during asexual parasite growth using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.