Role of the bicarbonate transporter SLC4γ in stony-coral skeleton formation and evolution.

Coral reefs are highly diverse ecosystems of immense ecological, economic, and aesthetic importance built on the calcium-carbonate-based skeletons of stony corals. The formation of these skeletons is threatened by increasing ocean temperatures and acidification, and a deeper understanding of the molecular mechanisms involved may assist efforts to mitigate the effects of such anthropogenic stressors. In this study, we focused on the role of the predicted bicarbonate transporter SLC4γ, which was suggested in previous studies to be a product of gene duplication and to have a role in coral-skeleton formation. Our comparative-genomics study using 30 coral species and 15 outgroups indicates that SLC4γ is present throughout the stony corals, but not in their non-skeleton-forming relatives, and apparently arose by gene duplication at the onset of stony-coral evolution. Our expression studies show that SLC4γ, but not the closely related and apparently ancestral SLC4ß, is highly upregulated during coral development coincident with the onset of skeleton deposition. Moreover, we show that juvenile coral polyps carrying CRISPR/Cas9-induced mutations in SLC4γ are defective in skeleton formation, with the severity of the defect in individual animals correlated with their frequencies of SLC4γ mutations. Taken together, the results suggest that the evolution of the stony corals involved the neofunctionalization of the newly arisen SLC4γ for a unique role in the provision of concentrated bicarbonate for calcium-carbonate deposition. The results also demonstrate the feasibility of reverse-genetic studies of ecologically important traits in adult corals.

Conversion of oxybenzone sunscreen to phototoxic glucoside conjugates by sea anemones and corals.

The reported toxicity of oxybenzone-based sunscreens to corals has raised concerns about the impacts of ecotourist-shed sunscreens on corals already weakened by global stressors. However, oxybenzone's toxicity mechanism(s) are not understood, hampering development of safer sunscreens. We found that oxybenzone caused high mortality of a sea anemone under simulated sunlight including ultraviolet (UV) radiation (290 to 370 nanometers). Although oxybenzone itself protected against UV-induced photo-oxidation, both the anemone and a mushroom coral formed oxybenzone-glucoside conjugates that were strong photo-oxidants. Algal symbionts sequestered these conjugates, and mortality correlated with conjugate concentrations in animal cytoplasm. Higher mortality in anemones that lacked symbionts suggests an enhanced risk from oxybenzone to corals bleached by rising temperatures. Because many commercial sunscreens contain structurally related chemicals, understanding metabolite phototoxicity should facilitate the development of coral-safe products.

Impact of Menthol on Growth and Photosynthetic Function of Breviolum Minutum (Dinoflagellata, Dinophyceae, Symbiodiniaceae) and Interactions with its Aiptasia Host.

Environmental change, including global warming and chemical pollution, can compromise cnidarian-(e.g., coral-) dinoflagellate symbioses and cause coral bleaching. Understanding the mechanisms that regulate these symbioses will inform strategies for sustaining healthy coral-reef communities. A model system for corals is the symbiosis between the sea anemone Exaiptasia pallida (common name Aiptasia) and its dinoflagellate partners (family Symbiodiniaceae). To complement existing studies of the interactions between these organisms, we examined the impact of menthol, a reagent often used to render cnidarians aposymbiotic, on the dinoflagellate Breviolum minutum, both in culture and in hospite. In both environments, the growth and photosynthesis of this alga were compromised at either 100 or 300 µM menthol. We observed reduction in PSII and PSI functions, the abundances of reaction-center proteins, and, at 300 µM menthol, of total cellular proteins. Interestingly, for free-living algae exposed to 100 µM menthol, an initial decline in growth, photosynthetic activities, pigmentation, and protein abundances reversed after 5-15 d, eventually approaching control levels. This behavior was observed in cells maintained in continuous light, but not in cells experiencing a light-dark regimen, suggesting that B. minutum can detoxify menthol or acclimate and repair damaged photosynthetic complexes in a light- and/or energy-dependent manner. Extended exposures of cultured algae to 300 µM menthol ultimately resulted in algal death. Most symbiotic anemones were also unable to survive this menthol concentration for 30 d. Additionally, cells impaired for photosynthesis by pre-treatment with 300 µM menthol exhibited reduced efficiency in re-populating the anemone host.

Proteasome Activity Is Influenced by the HECT_2 Protein Ipa1 in Budding Yeast.

The ubiquitin-proteasome system (UPS) controls cellular functions by maintenance of a functional proteome and degradation of key regulatory proteins. Central to the UPS is the proteasome that adjusts the abundance of numerous proteins, thereby safeguarding their activity or initiating regulatory events. Here, we demonstrate that the essential Saccharomyces cerevisiae protein Yjr141w/Ipa1 (Important for cleavage and PolyAdenylation) belongs to the HECT_2 (homologous to E6-AP carboxyl terminus_2) family. We found that five cysteine residues within the HECT_2 family signature and the C-terminus are essential for Ipa1 activity. Furthermore, Ipa1 interacts with several ubiquitin-conjugating enzymes in vivo and localizes to the cytosol and nucleus. Importantly, Ipa1 has an impact on proteasome activity, which is indicated by the activation of the Rpn4 regulon as well as by decreased turnover of destabilized proteasome substrates in an IPA1 mutant. These changes in proteasome activity might be connected to reduced maturation or modification of proteasomal core particle proteins. Our results highlight the influence of Ipa1 on the UPS. The conservation within the HECT_2 family and the connection of the human HECT_2 family member to an age-related degeneration disease might suggest that HECT_2 family members share a conserved function linked to proteasome activity.

The Mitotic Exit Network Regulates Spindle Pole Body Selection During Sporulation of Saccharomyces cerevisiae.

Age-based inheritance of centrosomes in eukaryotic cells is associated with faithful chromosome distribution in asymmetric cell divisions. During Saccharomyces cerevisiae ascospore formation, such an inheritance mechanism targets the yeast centrosome equivalents, the spindle pole bodies (SPBs) at meiosis II onset. Decreased nutrient availability causes initiation of spore formation at only the younger SPBs and their associated genomes. This mechanism ensures encapsulation of nonsister genomes, which preserves genetic diversity and provides a fitness advantage at the population level. Here, by usage of an enhanced system for sporulation-induced protein depletion, we demonstrate that the core mitotic exit network (MEN) is involved in age-based SPB selection. Moreover, efficient genome inheritance requires Dbf2/20-Mob1 during a late step in spore maturation. We provide evidence that the meiotic functions of the MEN are more complex than previously thought. In contrast to mitosis, completion of the meiotic divisions does not strictly rely on the MEN whereas its activity is required at different time points during spore development. This is reminiscent of vegetative MEN functions in spindle polarity establishment, mitotic exit, and cytokinesis. In summary, our investigation contributes to the understanding of age-based SPB inheritance during sporulation of S. cerevisiae and provides general insights on network plasticity in the context of a specialized developmental program. Moreover, the improved system for a developmental-specific tool to induce protein depletion will be useful in other biological contexts.

Controlling Protein Activity and Degradation Using Blue Light.

Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light).

Biophotography: concepts, applications and perspectives.

Synthetic biology aims at manipulating biological systems by rationally designed and genetically introduced components. Efforts in photoactuator engineering resulted in microorganisms reacting to extracellular light-cues with various cellular responses. Some of them lead to the formation of macroscopically observable outputs, which can be used to generate images made of living matter. Several methods have been developed to convert colorless compounds into visible pigments by an enzymatic conversion. This has been exploited as a showcase for successful creation of an optogenetic tool; examples for basic light-controlled biological processes that have been coupled to this biophotography comprise regulation of transcription, protein stability, and second messenger synthesis. Moreover, biological reproduction of images is used as means to facilitate quantitative characterization of optogenetic switches as well as a technique to investigate complex cellular signaling circuits. Here, we will compare the different techniques for biological image generation, introduce experimental approaches, and provide future-perspectives for biophotography.

The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion.

Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery.

A tobacco etch virus protease with increased substrate tolerance at the P1' position.

Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein-protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1' Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1' Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.

A LOV2 domain-based optogenetic tool to control protein degradation and cellular function.

Light perception is indispensable for plants to respond adequately to external cues and is linked to proteolysis of key transcriptional regulators. To provide synthetic light control of protein stability, we developed a generic photosensitive degron (psd) module combining the light-reactive LOV2 domain of Arabidopsis thaliana phot1 with the murine ornithine decarboxylase-like degradation sequence cODC1. Functionality of the psd module was demonstrated in the model organism Saccharomyces cerevisiae. Generation of conditional mutants, light regulation of cyclin-dependent kinase activity, light-based patterning of cell growth, and yeast photography exemplified its versatility. In silico modeling of psd module behavior increased understanding of its characteristics. This engineered degron module transfers the principle of light-regulated degradation to nonplant organisms. It will be highly beneficial to control protein levels in biotechnological or biomedical applications and offers the potential to render a plethora of biological processes light-switchable.

Targeted protein depletion in Saccharomyces cerevisiae by activation of a bidirectional degron.

BACKGROUND: Tools for in vivo manipulation of protein abundance or activity are highly beneficial for life science research. Protein stability can be efficiently controlled by conditional degrons, which induce target protein degradation at restrictive conditions. RESULTS: We used the yeast Saccharomyces cerevisiae for development of a conditional, bidirectional degron to control protein stability, which can be fused to the target protein N-terminally, C-terminally or placed internally. Activation of the degron is achieved by cleavage with the tobacco etch virus (TEV) protease, resulting in quick proteolysis of the target protein. We found similar degradation rates of soluble substrates using destabilization by the N- or C-degron. C-terminal tagging of essential yeast proteins with the bidirectional degron resulted in deletion-like phenotypes at non-permissive conditions. Developmental process-specific mutants were created by N- or C-terminal tagging of essential proteins with the bidirectional degron in combination with sporulation-specific production of the TEV protease. CONCLUSIONS: We developed a system to influence protein abundance and activity genetically, which can be used to create conditional mutants, to regulate the fate of single protein domains or to design artificial regulatory circuits. Thus, this method enhances the toolbox to manipulate proteins in systems biology approaches considerably.