C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways.

Protein quality control (PQC) mechanisms are essential for degradation of misfolded or dysfunctional proteins. An essential part of protein homeostasis is recognition of defective proteins by PQC components and their elimination by the ubiquitin-proteasome system, often concentrating on protein termini as indicators of protein integrity. Changes in amino acid composition of C-terminal ends arise through protein disintegration, alternative splicing, or during the translation step of protein synthesis from premature termination or translational stop-codon read-through. We characterized reporter protein stability using light-controlled exposure of the random C-terminal peptide collection (CtPC) in budding yeast revealing stabilizing and destabilizing features of amino acids at positions -5 to -1 of the C terminus. The (de)stabilization properties of CtPC-degrons depend on amino acid identity, position, as well as composition of the C-terminal sequence and are transferable. Evolutionary pressure toward stable proteins in yeast is evidenced by amino acid residues under-represented in cytosolic and nuclear proteins at corresponding C-terminal positions, but over-represented in unstable CtPC-degrons, and vice versa. Furthermore, analysis of translational stop-codon read-through peptides suggested that such extended proteins have destabilizing C termini. PQC pathways targeting CtPC-degrons involved the ubiquitin-protein ligase Doa10 and the cullin-RING E3 ligase SCFDas1 (Skp1-Cullin-F-box protein). Overall, our data suggest a proteome protection mechanism that targets proteins with unnatural C termini by recognizing a surprisingly large number of C-terminal sequence variants.

Optogenetic control of Cdc48 for dynamic metabolic engineering in yeast.

Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid ß-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.

Light-induced fermenter production of derivatives of the sweet protein monellin is maximized in prestationary Saccharomyces cerevisiae cultures.

Optogenetics has great potential for biotechnology and metabolic engineering due to the cost-effective control of cellular activities. The usage of optogenetics techniques for the biosynthesis of bioactive molecules ensures reduced costs and enhanced regulatory possibilities. This requires development of efficient methods for light-delivery during a production process in a fermenter. Here, we benchmarked the fermenter production of a low-caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. An expression system based on the light-controlled interaction between Cry2 and Cib1 was used for sweet-protein production. Optimization of the fermenter process was achieved by increasing the light-flux during the production phase to circumvent shading by yeast cells at high densities. Maximal amounts of the sweet-protein were produced in a pre-stationary growth phase, whereas at later stages, a decay in protein abundance was observable. Our investigation showcases the upscaling of an optogenetic production process from small flasks to a bioreactor. Optogenetic-controlled production in a fermenter is highly cost-effective due to the cheap inducer and therefore a viable alternative to chemicals for a process that requires an induction step.

An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance.

Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

An Optogenetic Tool for Induced Protein Stabilization Based on the Phaeodactylum tricornutum Aureochrome 1a Light-Oxygen-Voltage Domain.

Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light-oxygen-voltage (LOV) domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254, and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark-state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic adenosine monophosphate production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.

Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum-associated degradation pathway.

Protein quality mechanisms are fundamental for proteostasis of eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) is a well-studied pathway that ensures quality control of secretory and endoplasmic reticulum (ER)-resident proteins. Different branches of ERAD are involved in degradation of malfolded secretory proteins, depending on the localization of the misfolded part, the ER lumen (ERAD-L), the ER membrane (ERAD-M), and the cytosol (ERAD-C). Here we report that modification of several ER transmembrane proteins with the photosensitive degron (psd) module resulted in light-dependent degradation of the membrane proteins via the ERAD-C pathway. We found dependency on the ubiquitylation machinery including the ubiquitin-activating enzyme Uba1, the ubiquitin--conjugating enzymes Ubc6 and Ubc7, and the ubiquitin-protein ligase Doa10. Moreover, we found involvement of the Cdc48 AAA-ATPase complex members Ufd1 and Npl4, as well as the proteasome, in degradation of Sec62-myc-psd. Thus, our work shows that ERAD-C substrates can be systematically generated via synthetic degron constructs, which facilitates future investigations of the ERAD-C pathway.

Optogenetic Downregulation of Protein Levels with an Ultrasensitive Switch.

Optogenetic control of protein activity is a versatile technique to gain control over cellular processes, for example, for biomedical and biotechnological applications. Among other techniques, the regulation of protein abundance by controlling either transcription or protein stability found common use as this controls the activity of any type of target protein. Here, we report modules of an improved variant of the photosensitive degron module and a light-sensitive transcription factor, which we compared to doxycycline-dependent transcriptional control. Given their modularity the combined control of synthesis and stability of a given target protein resulted in the synergistic down regulation of its abundance by light. This combined module exhibits very high switching ratios, profound downregulation of protein abundance at low light-fluxes, and fast protein depletion kinetics. Overall, this synergistic optogenetic multistep control (SOMCo) module is easy to implement and results in a regulation of protein abundance superior to each individual component.

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.

Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light.

In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.

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.

Photo-sensitive degron variants for tuning protein stability by light.

BACKGROUND: Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus. RESULTS: We used the model organism Saccharomyces cerevisiae to generate psd module variants with increased and decreased stabilities in darkness or when exposed to blue light using site-specific and random mutagenesis. The variants were characterized as fusions to fluorescent reporter proteins and showed half-lives between 6 and 75 minutes in cells exposed to blue light and 14 to 187 minutes in darkness. In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module. Measuring the dark/light ratio of selected constructs in yeast cells showed that two variants were obtained with ratios twice as high as in the wild type psd module. In silico modeling of photoreceptor variant characteristics suggested that for most cases alterations in behavior were induced by changes in the light-response of the LOV2 domain. CONCLUSIONS: In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light. Two variants showed characteristics that are profoundly improved compared to the original construct. The modular usage of the LOV2 domain in optogenetic tools allows the usage of the mutants in the context of other applications in synthetic and systems biology as well.

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.

Acetate regulation of spore formation is under the control of the Ras/cyclic AMP/protein kinase A pathway and carbon dioxide in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2Δ) or carbonic anhydrase (nce103Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.

TIPI: TEV protease-mediated induction of protein instability.

Reverse genetics approaches require methods to inactivate a specific protein. One possibility is to modify the target protein with a degradation signal (degron). Degrons are short, transferable sequences that confer protein instability. They target proteins for degradation either constitutively or after activation, e.g., by phosphorylation, presence of a binding partner, or conformational rearrangements in the substrate. In this chapter, we describe a synthetic way to activate a degron. It employs the generation of an N-degron by cleavage of a substrate with the site-specific tobacco etch virus (TEV) protease. Subsequently, the substrate is targeted for degradation by the ubiquitin-proteasome system. This TEV protease-induced protein instability system provides a powerful approach to generate conditional mutants for synthetic biology or for the investigation of protein functions in a specific cellular context.

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.