Development of AlissAID system targeting GFP or mCherry fusion protein.

Conditional control of target proteins using the auxin-inducible degron (AID) system provides a powerful tool for investigating protein function in eukaryotes. Here, we established an Affinity-linker based super-sensitive auxin-inducible degron (AlissAID) system in budding yeast by using a single domain antibody (a nanobody). In this system, target proteins fused with GFP or mCherry were degraded depending on a synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA). In AlissAID system, nanomolar concentration of 5-Ad-IAA induces target degradation, thus minimizing the side effects from chemical compounds. In addition, in AlissAID system, we observed few basal degradations which was observed in other AID systems including ssAID system. Furthermore, AlissAID based conditional knockdown cell lines are easily generated by using budding yeast GFP Clone Collection. Target protein, which has antigen recognition sites exposed in cytosol or nucleus, can be degraded by the AlissAID system. From these advantages, the AlissAID system would be an ideal protein-knockdown system in budding yeast cells.

Regulator of Awn Elongation 3, an E3 ubiquitin ligase, is responsible for loss of awns during African rice domestication.

Two species of rice have been independently domesticated from different ancestral wild species in Asia and Africa. Comparison of mutations that underlie phenotypic and physiological alterations associated with domestication traits in these species gives insights into the domestication history of rice in both regions. Asian cultivated rice, Oryza sativa, and African cultivated rice, Oryza glaberrima, have been modified and improved for common traits beneficial for humans, including erect plant architecture, nonshattering seeds, nonpigmented pericarp, and lack of awns. Independent mutations in orthologous genes associated with these traits have been documented in the two cultivated species. Contrary to this prevailing model, selection for awnlessness targeted different genes in O. sativa and O. glaberrima. We identify Regulator of Awn Elongation 3 (RAE3) a gene that encodes an E3 ubiquitin ligase and is responsible for the awnless phenotype only in O. glaberrima. A 48-bp deletion may disrupt the substrate recognition domain in RAE3 and diminish awn elongation. Sequencing analysis demonstrated low nucleotide diversity in a ~600-kb region around the derived rae3 allele on chromosome 6 in O. glaberrima compared with its wild progenitor. Identification of RAE3 sheds light on the molecular mechanism underlying awn development and provides an example of how selection on different genes can confer the same domestication phenotype in Asian and African rice.

The Ubiquitin Ligase SCF(Ucc1) Acts as a Metabolic Switch for the Glyoxylate Cycle.

Despite the crucial role played by the glyoxylate cycle in the virulence of pathogens, seed germination in plants, and sexual development in fungi, we still have much to learn about its regulation. Here, we show that a previously uncharacterized SCF(Ucc1) ubiquitin ligase mediates proteasomal degradation of citrate synthase in the glyoxylate cycle to maintain metabolic homeostasis in glucose-grown cells. Conversely, transcription of the F box subunit Ucc1 is downregulated in C2-compound-grown cells, which require increased metabolic flux for gluconeogenesis. Moreover, in vitro analysis demonstrates that oxaloacetate regenerated through the glyoxylate cycle induces a conformational change in citrate synthase and inhibits its recognition and ubiquitination by SCF(Ucc1), suggesting the existence of an oxaloacetate-dependent positive feedback loop that stabilizes citrate synthase. We propose that SCF(Ucc1)-mediated regulation of citrate synthase acts as a metabolic switch for the glyoxylate cycle in response to changes in carbon source, thereby ensuring metabolic versatility and flexibility.

Triacylglycerol lipase Tgl4 is a stable protein and its dephosphorylation is regulated in a cell cycle-dependent manner in Saccharomyces cerevisiae.

Triacylglycerols (TGs) serve as reservoirs for diacylglycerols and fatty acids, which play important roles in synthesizing energy and membrane lipids that are required for cell cycle progression. In the yeast, Saccharomyces cerevisiae, Tgl4, the functional ortholog of murine adipose triacylglycerol lipase (ATGL), is activated by Cdk1/Cdc28-mediated phosphorylation and facilitates the G1/S transition. However, little is known about how Tgl4 is inactivated during the cell cycle. To monitor the phosphorylation status and the stability of endogenous Tgl4, we raised a specific antibody against Tgl4. We found that in contrast to the previous suggestion, Tgl4 was a stable protein throughout the cell cycle. We also showed that Tgl4 was dephosphorylated upon entry into G1 phase. These results suggest that Tgl4 is a stable protein and is inactivated during G1 phase by dephosphorylation.

A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.

Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed. One substrate is Sec61-2, a temperature sensitive allele of the Sec61 translocation channel. Cells expressing Sec61-2 grow at 25 °C because the protein is stable, but sec61-2 yeast are inviable at 38 °C because the mutated protein is degraded in a Hrd1-dependent manner. Therefore, deleting HRD1 stabilizes Sec61-2 and hence sec61-2hrd1∆ double mutants are viable at 38 °C. This unique phenotype allowed us to perform a non-biased screen for loss-of-function alleles in HRD1. Based on its importance in mediating substrate retrotranslocation, the screen was also developed to focus on mutations in sequences encoding Hrd1's transmembrane-rich domain. Ultimately, a group of recessive mutations was identified in HRD1, including an ensemble of destabilizing mutations that resulted in the delivery of Hrd1 to the ERAD pathway. A more stable mutant resided in a buried transmembrane domain, yet the Hrd1 complex was disrupted in yeast expressing this mutant. Together, these data confirm the importance of Hrd1 complex integrity during ERAD, suggest that allosteric interactions between transmembrane domains regulate Hrd1 complex formation, and provide the field with new tools to define the dynamic interactions between ERAD components during substrate retrotranslocation.

A tonoplast-localized magnesium transporter is crucial for stomatal opening in Arabidopsis under high Mg2+ conditions.

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing under changing environmental conditions are still not completely understood. Through large-scale genetic screening, we isolated an Arabidopsis mutant (closed stomata2 (cst2)) that is defective in stomatal opening. We cloned the causal gene (MGR1/CST2) and functionally characterized this gene. The mutant phenotype was caused by a mutation in a gene encoding an unknown protein with similarities to the human magnesium (Mg2+ ) efflux transporter ACDP/CNNM. MGR1/CST2 was localized to the tonoplast and showed transport activity for Mg2+ . This protein was constitutively and highly expressed in guard cells. Knockout of this gene resulted in stomatal closing, decreased photosynthesis and growth retardation, especially under high Mg2+ conditions, while overexpression of this gene increased stomatal opening and tolerance to high Mg2+ concentrations. Furthermore, guard cell-specific expression of MGR1/CST2 in the mutant partially restored its stomatal opening. Our results indicate that MGR1/CST2 expression in the leaf guard cells plays an important role in maintaining cytosolic Mg2+ concentrations through sequestering Mg2+ into vacuoles, which is required for stomatal opening, especially under high Mg2+ conditions.

A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair.

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

The Rim101 pathway mediates adaptation to external alkalization and altered lipid asymmetry: hypothesis describing the detection of distinct stresses by the Rim21 sensor protein.

Yeast cells adapt to alkaline conditions by activating the Rim101 alkali-responsive pathway. Rim21 acts as a sensor in the Rim101 pathway and detects extracellular alkalization. Interestingly, Rim21 is also known to be activated by alterations involving the lipid asymmetry of the plasma membrane. In this study, we briefly summarize the mechanism of activation and the signal transduction cascade of the Rim101 pathway and propose a hypothesis on how Rim21 is able to detect distinct signals, particularly external alkalization, and altered lipid asymmetry. We found that external alkalization can suppress transbilayer movements of phospholipids between the two leaflets of the plasma membrane, which may lead to the disturbance of the lipid asymmetry of the plasma membrane. Therefore, we propose that external alteration is at least partly sensed by Rim21 through alterations in lipid asymmetry. Understanding this activation mechanism could greatly contribute to drug development against fungal infections.

Rapid turnover of transcription factor Rim101 confirms a flexible adaptation mechanism against environmental stress in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells activate the Rim101 pathway to adapt to alkaline and salt stresses. On activation of this pathway, the transcription factor Rim101 undergoes proteolytic activation and regulates the expression of responsive genes. We found Rim101 to be a short-lived protein with a half-life of approximately 15 min. Its rapid turnover was supposedly mediated by the ubiquitin-proteasome system. Excess accumulation of the processed active Rim101 through its over-expression conferred tolerance to both alkaline and salt stresses in yeast cells; in contrast, it had detrimental effects under cadmium stress condition. Cadmium ion inhibited proteolytic activation of Rim101, implying reciprocal interaction between the Rim101 pathway and cadmium stress. Our results showed yeast cells to be equipped with two protective systems to prevent overaccumulation of the processed active Rim101; Rim101 processing is inhibited when Rim101 level is high, and turnover of processed Rim101 is accelerated when it is abundant. Collectively, the results confirmed the flexible aspect of stress response in yeast cell; the cells not only prevent excess activation of one stress-responsive pathway but also facilitate its attenuation to cope with other environmental stresses.

N-glycosylation of Rim21 at an Unconventional Site Fine-tunes Its Behavior in the Plasma Membrane.

The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.

Insulin-like growth factor 1 receptor stabilizes the ETV6-NTRK3 chimeric oncoprotein by blocking its KPC1/Rnf123-mediated proteasomal degradation.

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.

Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice.

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

The nutrient stress-induced small GTPase Rab5 contributes to the activation of vesicle trafficking and vacuolar activity.

Rab family small GTPases regulate membrane trafficking by spatiotemporal recruitment of various effectors. However, it remains largely unclear how the expression and functions of Rab proteins are regulated in response to extracellular or intracellular stimuli. Here we show that Ypt53, one isoform of Rab5 in Saccharomyces cerevisiae, is up-regulated significantly under nutrient stress. Under non-stress conditions, Vps21, a constitutively expressed Rab5 isoform, is crucial to Golgi-vacuole trafficking and to vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, the up-regulated Ypt53 and the constitutive Vps21 function redundantly to maintain these activities, which, in turn, prevent the accumulation of reactive oxygen species and maintain mitochondrial respiration. Together, our results clarify the relative roles of these constitutive and nutrient stress-inducible Rab5 proteins that ensure adaptable vesicle trafficking and vacuolar hydrolase activity, thereby allowing cells to adapt to environmental changes.

Activation of double-stranded RNA-activated protein kinase (PKR) by interferon-stimulated gene 15 (ISG15) modification down-regulates protein translation.

The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2α to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2α to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.

E3 Ligases Regulate Organelle Inheritance in Yeast.

Saccharomyces cerevisiae proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin-myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.

Role of the San1 ubiquitin ligase in the heat stress-induced degradation of nonnative Nup1 in the nuclear pore complex.

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system. The ubiquitin-proteasome system may eliminate aberrant, misfolded, and/or orphan components; however, the involvement of the ubiquitin-proteasome system in the degradation of nonnative Nups in the NPC remains unclear. Here, we show that in Saccharomyces cerevisiae, although Nup1 (the FG-Nup component of the central core of the NPC) was stable, C-terminally green fluorescent protein-tagged Nup1, which had been incorporated into the NPC, was degraded by the proteasome especially under heat stress conditions. The degradation was dependent on the San1 ubiquitin ligase and Cdc48/p97, as well as its cofactor Doa1. We also demonstrate that San1 weakly but certainly contributes to the degradation of nontagged endogenous Nup1 in cells defective in NPC biogenesis by the deletion of NUP120. In addition, the overexpression of SAN1 exacerbated the growth defect phenotype of nup120Δ cells, which may be caused by excess degradation of defective Nups due to the deletion of NUP120. These biochemical and genetic data suggest that San1 is involved in the degradation of nonnative Nups generated by genetic mutation or when NPC biogenesis is impaired.

A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.

Targeted Protein Degradation Systems: Controlling Protein Stability Using E3 Ubiquitin Ligases in Eukaryotic Species.

This review explores various methods for modulating protein stability to achieve target protein degradation, which is a crucial aspect in the study of biological processes and drug design. Thirty years have passed since the introduction of heat-inducible degron cells utilizing the N-end rule, and methods for controlling protein stability using the ubiquitin-proteasome system have moved from academia to industry. This review covers protein stability control methods, from the early days to recent advancements, and discusses the evolution of techniques in this field. This review also addresses the challenges and future directions of protein stability control techniques by tracing their development from the inception of protein stability control methods to the present day.