Yeast Protein Research Articles

A ribosome-associating chaperone mediates GTP-driven vectorial folding of nascent eEF1A

Eukaryotic translation elongation factor 1A (eEF1A) is a highly abundant, multi-domain GTPase. Post-translational steps essential for eEF1A biogenesis are carried out by bespoke chaperones but co-translational mechanisms tailored to eEF1A folding remain unexplored. Here, we use AlphaPulldown to identify Ypl225w (also known as Chp1, Chaperone 1 for eEF1A) as a conserved yeast protein predicted to stabilize the N-terminal, GTP-binding (G) domain of eEF1A against its misfolding propensity, as predicted by computational simulations and validated by microscopy analysis of ypl225wΔ cells. Proteomics and biochemical reconstitution reveal that Ypl225w functions as a co-translational chaperone by forming dual interactions with the eEF1A G domain nascent chain and the UBA domain of ribosome-bound nascent polypeptide-associated complex (NAC). Lastly, we show that Ypl225w primes eEF1A nascent chains for binding to GTP as part of a folding mechanism tightly coupled to chaperone recycling. Our work shows that an ATP-independent chaperone can drive vectorial folding of nascent chains by co-opting G protein nucleotide binding.

Expression and purification of PNGase F protein in yeast and its anti-PRV activity.

Pseudorabies virus (Pseudorabiesvirus, PRV) has caused huge economic losses to the global pig industry. In recent years, it has been reported that there are PRV mutants, but the traditional vaccine can not completely prevent or control the infection of PRV, so there is an urgent need to develop new broad-spectrum anti-disease drugs for prevention and treatment. PNGase F from bacteria can catalyze the hydrolysis of oligosaccharides linked to asparagine residues on peptides, so we speculate that PNGase F can inhibit virus infection by removing the glycosylation of virus membrane glycoproteins. In this study, PNGase F protein was highly expressed and purified in Pichia pastoris, and the deglycosylation activity of PNGase F expressed in Pichia pastoris was verified. In vitro, 15μM could significantly inhibit the proliferation of virus in cells. The results of cytotoxicity test showed that PNGase F was not toxic to many cells. To further evaluate the effect of PNGase F in different stages of virus infection, it was found that PNGase F had significant inhibitory effect on virus adsorption and invasion. In vivo experiments in mice, PNGase F could significantly inhibit the replication of PRV Ea strain in mice and inhibit PRV, reduced brain lesions. Our experiments show that PNGase F expressed by yeast can inhibit PRV infection in vitro and in vitro, and its inhibitory mechanism is preliminarily discussed, which can provide a new reference for the development of broad-spectrum antiviral drugs based on PNGase F.

Long Noncoding RNAs Responding to Ethanol Stress in Yeast Seem Associated with Protein Synthesis and Membrane Integrity

Background/Objectives: Translation and the formation of membraneless organelles are linked mechanisms to promote cell stress surveillance. LncRNAs responsive to ethanol stress transcr_9136 of the SEY6210 strain and transcr_10027 of the BY4742 strain appear to act on tolerance to ethanol in these strains. Here, we investigate whether the ethanol responsiveness of transcr_9136 and transcr_10027 and their role in ethanol stress are associated with protein biogenesis and membraneless organelle assembly. Methods: SEY6210 transcr_9136∆ and BY4742 transcr_10027∆ and their wild-type counterparts were subjected to their maximum ethanol-tolerant stress. The expression of the transcr_9136, transcr_10027, ILT1, RRP1, 27S, 25S, TIR3, and FAA3 genes was accessed by qPCR. The level of DCP1a, PABP, and eIF4E proteins was evaluated by Western blotting. Bioinformatics analyses allowed us to check whether transcr_9136 may regulate the expression of RRP1 and predict the interaction between transcr_10027 and Tel1p. The cell death rate of SEY6210 strains under control and ethanol stress conditions was assessed by flow cytometry. Finally, we evaluated the total protein yield of all strains analyzed. Results: The results demonstrated that transcr_9136 of SEY6210 seems to control the expression of RRP1 and 27S rRNA and reduce the general translation. Furthermore, transcr_9136 seems to act on cell membrane integrity. Transcr_10027 of BY4742 appears to inhibit processing body formation and induce a general translation level. Conclusions: This is the first report on the effect of lncRNAs on yeast protein synthesis and new mechanisms of stress-responsive lncRNAs in yeast, with potential industrial applications such as ethanol production.

Screening a 681-membered yeast collection for the secretion of proteins with antifungal activity.

Fungal pathogens pose a threat to human health and food security. Few antifungals are available and resistance to all has been reported. Novel strategies to control plant and human pathogens as well as food spoilers are urgently required. Environmental yeasts provide a functionally diverse, yet underexploited potential for fungal control based on their natural competition via the secretion of proteins and other small molecules such as iron chelators, volatile organic compounds or biosurfactants. However, there is a lack of standardized workflows to systematically access application-relevant yeast-based compounds and understand their molecular functioning. Towards this goal, we developed a workflow to identify and characterize yeast isolates that are active against spoilage yeasts and relevant human and plant pathogens, herein focusing on discovering yeasts that secrete antifungal proteins. The workflow includes the classification of the secreted molecules and cross-comparison of their antifungal capacity using an independent synthetic calibrant. Our workflow delivered a collection of 681 yeasts of which 212 isolates (31%) displayed antagonism against at least one target strain. While 57.5% of the active yeasts showed iron-depended antagonism, likely due to pulcherrimin-like iron chelators, 31.7% secreted antifungal proteins. Those yeast candidates clustered within twelve OTUs, showed narrow and broad target spectra, and several showed a broad pH and temperature activity profile. Given the tools for yeast biotechnology and protein engineering available, our collection can serve as a rich starting point for genetic and molecular characterization of the various antifungal phenotypes, their mode of action and their future exploitation.

The Asgard archaeal origins of Arf family GTPases involved in eukaryotic organelle dynamics.

The evolution of eukaryotes is a fundamental event in the history of life. The closest prokaryotic lineage to eukaryotes, the Asgardarchaeota, encode proteins previously found only in eukaryotes, providing insight into their archaeal ancestor. Eukaryotic cells are characterized by endomembrane organelles, and the Arf family GTPases regulate organelle dynamics by recruiting effector proteins to membranes upon activation. The Arf family is ubiquitous among eukaryotes, but its origins remain elusive. Here we report a group of prokaryotic GTPases, the ArfRs, which are widely present in Asgardarchaeota. Phylogenetic analyses reveal that eukaryotic Arf family proteins arose from the ArfR group. Expression of representative Asgardarchaeota ArfR proteins in yeast and X-ray crystallographic studies show that ArfR GTPases possess the mechanism of membrane binding and structural features unique to Arf family proteins. Our results indicate that Arf family GTPases originated in the archaeal ancestor of eukaryotes, consistent with aspects of the endomembrane system evolving early in eukaryogenesis.

Rad5 and Ubc4 directly ubiquitinate PCNA at Lys164 in vitro.

Ubiquitination of the proliferating cell nuclear antigen (PCNA) by the budding yeast protein Rad5 have important functions in replication stress responses. Rad5 together with the Ubc13-Mms2 complex attaches Lys63-linked ubiquitin chain to a highly conserved Lys164 residue in PCNA. The reaction requires prior PCNA mono-ubiquitination by the Rad6-Rad18 complex and signals for error-free DNA damage tolerance responses. Cellular studies suggested that Rad5 also cooperates with Ubc4 to catalyze PCNA ubiquitination in response to Okazaki fragment ligation defects, but biochemical evidence of this reaction is lacking. Here we reconstituted this reaction and studied its biochemical properties. We found that Rad5 and Ubc4 directly ubiquitinate PCNA and the reaction requires a coordination of Rad5's HIRAN and RING domains. Most interestingly, we found that the reaction ubiquitinates PCNA at multiple sites among which Lys164 is a major ubiquitination site. These findings suggest that Rad5 may contribute to replication stress responses through a novel mechanism by directly ubiquitinating Lys164 in PCNA.

Fission yeast GPI inositol deacylase Bst1 regulates ER-Golgi transport and functions in late stages of cytokinesis.

The Munc13/UNC-13 family protein Ync13 is essential for septum integrity and cytokinesis in fission yeast. To further explore the mechanism of Ync13 functions, spontaneous suppressors of ync13 mutants, which can suppress the colony-formation defects and lysis phenotype of ync13 mutant cells, are isolated and characterized. One of the suppressor mutants, bst1-s27, shows defects in the cytokinetic contractile ring constriction, septation, and daughter-cell separation, similar to bst1Δ mutant. Bst1, a predicted GPI inositol deacylase, was an uncharacterized protein in fission yeast. It localizes to the nuclear ER and puncta structures in the cytoplasm. The Bst1 puncta overlaps frequently with Anp1, which is a marker of ER-Golgi transport, but rarely with trans-Golgi marker Sec72. The nuclear ER signal of Anp1 increases in bst1Δ mutant, whereas Sec72 localization shows no obvious changes. In addition, more cytoplasmic puncta structures of COPII subunits, Sec13 and Sec24, are observed in bst1Δ mutant, and acid phosphatase secretion is compromised without Bst1. Consistently, the division site targeting of the β-glucanase Eng1 and α-glucanase Agn1 is reduced in bst1Δ and bst1Δ ync13Δ mutant. Taken together, our results suggest that Bst1 regulates ER-Golgi transport and is involved in cytokinesis through regulating the secretion of glucanases.

A protein interaction map of the myosin Myo2 reveals a role of Alo1 in mitochondrial inheritance in yeast.

Budding yeast cells multiply by asymmetric cell division. During this process, the cell organelles are transported by myosin motors along the actin cytoskeleton into the growing bud, while at the same time some organelles must be retained in the mother cell. The ordered partitioning of organelles depends on highly regulated binding of motor proteins to cargo membranes. To search for novel components involved in this process, we performed a protein fragment complementation screen using the cargo binding domain of Myo2, the major organelle transporter in yeast, as a bait and a genome-wide strain collection expressing yeast proteins as prey. One robust hit was Alo1, a poorly characterized D-arabinono-1,4-lactone oxidase located in the mitochondrial outer membrane. We found that mutants lacking Alo1 exhibit defects in mitochondrial morphology and inheritance. During oxidative stress dysfunctional mitochondria are immobilized in the mother in wild type cells. Intriguingly, overexpression of ALO1 restores bud-directed transport of mitochondria under these conditions. We propose that Alo1 supports the recruitment of Myo2 to mitochondria and its activity is particularly important under oxidative stress.

Improving Cellular Protein Content of Saccharomyces cerevisiae Based on Adaptive Evolution and Flow Cytometry-Aided High Throughput Screening.

Enhancing the protein content and production efficiency of Saccharomyces cerevisiae is crucial as an alternative protein source. This study screened nongenetically modified yeast strains with high protein content for food ingredient production and explored the underlying mechanisms. Yeast protein levels were found to correlate with RNA, leading to a high-throughput screening method using RNA fluorescence and flow cytometry. Four mutant libraries (∼200,000 cells) were generated through adaptive laboratory evolution in protein synthesis inhibitors, resulting in the high protein mutant content B1, with a protein content of 65.8 g/100 g dry cell weight in shake flasks. In a 45 L bioreactor using fed-batch fermentation with ethanol below 1.5 g/L, B1's protein content increased to 70.3 g/100 g dry cell weight, an 18.5% rise. Mannan and β-glucan levels in the cell wall decreased by 21.7 and 30.5%, potentially enhancing protein extraction for food production. Transcriptome analysis revealed that increased protein content results from down-regulating the cell cycle and meiosis-related genes. Validation of differentially expressed genes demonstrated that up-regulating SUT1 and down-regulating CNM67 are key for enhancing protein synthesis and accumulation. This study proposes a nongenetic screening method for high protein content S. cerevisiae strains, achieving the highest reported protein content.

Genome-wide conditional degron libraries for functional genomics.

Functional genomics with libraries of knockout alleles is limited to non-essential genes and convoluted by the potential accumulation of suppressor mutations in knockout backgrounds, which can lead to erroneous functional annotations. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for inducible degradation of AID-tagged proteins in the budding yeast Saccharomyces cerevisiae. First, we determined that N-terminal tagging is at least twice as likely to inadvertently impair protein function across the proteome. We thus constructed two libraries with over 5,600 essential and non-essential proteins fused at the C-terminus with an AID tag and an optional fluorescent protein. Approximately 90% of AID-tagged proteins were degraded in the presence of the auxin analog 5-Ph-IAA, with initial protein abundance and tag accessibility as limiting factors. Genome-wide screens for DNA damage response factors revealed a role for the glucose signaling factor GSF2 in resistance to hydroxyurea, highlighting how the AID libraries extend the yeast genetics toolbox.

Utilizing the Fungal Bicistronic System for Multi-Gene Expression to Generate Insect-Resistant and Herbicide-Tolerant Maize.

Developing simple and efficient multi-gene expression systems is crucial for multi-trait improvement or bioproduction in transgenic plants. In previous research, an IGG6-based bicistronic system from the nonpathogenic fungus Glarea lozoyensis efficiently expressed multiple enzyme proteins in yeast and maize, and the heterologous enzymes successfully performed their catalytic activity to reconstruct the biosynthetic pathway in the host organism. Unlike enzyme proteins, some heterologous functional proteins (such as insecticidal proteins) are dose-dependent and they need to express sufficient levels to perform their biological functions. It remains unclear whether the IGG6-based bicistronic system can achieve high expression of the functional proteins for practical applications in crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, vip3Aa and cry1Ab, were linked via IGG6 to form a bicistron, while two glyphosate resistance genes, gr79epsps and gat, served as monocistronic selectable marker genes. Regenerated maize plants were produced through genetic transformation. RNA and immunoblot analyses revealed that the vip3Aa-IGG6-cry1Ab bicistron was transcribed as a single transcript, which was then translated into two separate proteins. Notably, the transcription and translation of cry1Ab were significantly positively correlated with those of vip3Aa. Through ELISA and leaf bioassay, we identified two transgenic maize lines, VICGG-15 and VICGG-20, that exhibited high insecticidal activity against fall armyworm (FAW; Spodoptera frugiperda) and Asian corn borer (ACB; Ostrinia furnacalis), both of which had high expression of Vip3Aa and Cry1Ab proteins. Subsequent evaluations, including silk, ear, and field bioassays, as well as glyphosate tolerance assessments, indicated that the VICGG-15 plants displayed high resistance to FAW and ACB, and could tolerate up to 3600 g acid equivalent (a.e.) glyphosate per hectare without adversely affecting phenotype or yield. Our finding established that the IGG6-based bicistronic system can achieve high expression of functional proteins in maize, and it is a potential candidate for multi-gene assembly and expression in plants.

Metabolic Engineering of Komagataella phaffii for Xylose Utilization from Cellulosic Biomass.

Cellulosic biomass hydrolysates are rich in glucose and xylose, but most microorganisms, including Komagataella phaffii, are unable to utilize xylose effectively. To address this limitation, we engineered a K. phaffii strain optimized for xylose metabolism through the xylose oxidoreductase pathway and promoter optimization. A promoter library with varying strengths was used to fine-tune the expression levels of the XYL1, XYL2, and XYL3 genes, resulting in a strain with a strong promoter for XYL2 and weaker promoters for XYL1 and XYL3. This engineered strain exhibited superior growth, achieving 14 g cells/L and a maximal growth rate of 0.4 g cells/L-h in kenaf hydrolysate, outperforming a native strain by 17%. This study is the first to report the introduction of the xylose oxidoreductase pathway into K. phaffii, demonstrating its potential as an industrial platform for producing yeast protein and other products from cellulosic biomass.

Proteome-wide forced interactions reveal a functional map of cell-cycle phospho-regulation in S. cerevisiae

ABSTRACT Dynamic protein phosphorylation and dephosphorylation play an essential role in cell cycle progression. Kinases and phosphatases are generally highly conserved across eukaryotes, underlining their importance for post-translational regulation of substrate proteins. In recent years, advances in phospho-proteomics have shed light on protein phosphorylation dynamics throughout the cell cycle, and ongoing progress in bioinformatics has significantly improved annotation of specific phosphorylation events to a given kinase. However, the functional impact of individual phosphorylation events on cell cycle progression is often unclear. To address this question, we used the Synthetic Physical Interactions (SPI) method, which enables the systematic recruitment of phospho-regulators to most yeast proteins. Using this method, we identified several putative novel targets involved in chromosome segregation and cytokinesis. The SPI method monitors cell growth and, therefore, serves as a tool to determine the impact of protein phosphorylation on cell cycle progression.

Effects of Peptidase Treatment on Properties of Yeast Protein as an Alternative Protein Source.

Yeast protein, high-quality and high-content microbial protein, can serve as alternative sources of protein. This study examined the structural and functional characteristics of yeast protein through enzymatic treatment using different ratios of alcalase (endo-type) and prozyme 2000P (exo-type) including 2:1 (A2P1), 1:1 (A1P1), and 1:2 (A1P2). After enzymatic hydrolysis, a significant increase in protein solubility from less than 3.1% in untreated proteins to around 16%, particularly at pH 2 or pH 12. Furthermore, a maximum degree of hydrolysis of over 85% was achieved after enzyme treatment. Among them, the highest value of 87.73% was achieved at yeast protein treated by A1P2. Scanning electron microscopy images revealed varied surface morphologies, with exhibiting an increased surface area, particularly after treatment using A2P1. Next, yeast protein treated with A2P1 also demonstrated a superior emulsion stability index (3364.17). However, the antioxidant capacity was higher in proteins treated with A1P2 (78.30%). In addition, the elevated levels of certain amino acids, specifically leucine, lysine, phenylalanine, valine, and arginine, thereby indicating an enhanced amino acid profile was observed. Overall, yeast proteins treated with complex enzymes exhibited improved functionality and potential for diverse food applications.

ReCRAC: A Stringent Method for Precise Mapping of Protein-RNA Interactions in Yeast.

Intricate interactions between RNA-binding proteins (RBPs) and RNA play pivotal roles in cellular homeostasis, impacting a spectrum of biological processes vital for survival. UV crosslinking methods to study protein-RNA interactions have been instrumental in elucidating their interactions but can be limited by degradation of target proteins during the process, low signal-to-noise ratios, and nonspecific interactions. Addressing these limitations, we describe reCRAC (reverse CRAC), a novel adaptation of the CRAC (crosslinking and analysis of cDNA) technique, optimized for yeast Saccharomyces cerevisiae. Like CRAC, reCRAC applies tandem affinity purification to yield highly enriched protein preparations. However, reCRAC is redesigned to work with unstable proteins. This is achieved by lysing the cells directly into highly denaturing buffer conditions, followed by stringent purification steps. The reCRAC method was successfully applied to the easily degraded yeast protein Pin4, allowing identification of precise binding sites at base-pair resolution with greatly reduced target protein degradation and improved signal-to-noise ratios.

Bimolecular Fluorescence Complementation as a Tool to Study Specific Dynamic Interactions Between Proteins in Fission Yeast.

Bimolecular fluorescence complementation (BiFC) is a technique that enables real-time observation within living cells of the interaction between two proteins forming a complex, determining the location where such interaction occurs within the cell, and even the association and dissociation cycles in response to physiological cues. Here, we describe in detail the use of bimolecular fluorescence complementation to visualize the assembly and disassembly of cohesin over the fission yeast cell cycle.

Subtleties in Clathrin heavy chain binding boxes provide selectivity among adaptor proteins of budding yeast

Clathrin forms a triskelion, or three-legged, network that regulates cellular processes by facilitating cargo internalization and trafficking in eukaryotes. Its N-terminal domain is crucial for interacting with adaptor proteins, which link clathrin to the membrane and engage with specific cargo. The N-terminal domain contains up to four adaptor-binding sites, though their role in preferential occupancy by adaptor proteins remains unclear. In this study, we examine the binding hierarchy of adaptors for clathrin, using integrative biophysical and structural approaches, along with in vivo functional experiments. We find that yeast epsin Ent5 has the highest affinity for clathrin, highlighting its key role in cellular trafficking. Epsins Ent1 and Ent2, crucial for endocytosis but thought to have redundant functions, show distinct binding patterns. Ent1 exhibits stronger interactions with clathrin than Ent2, suggesting a functional divergence toward actin binding. These results offer molecular insights into adaptor protein selectivity, suggesting they competitively bind clathrin while also targeting three different clathrin sites.

A Semi-supervised Protein Complex Identification Algorithm Based on Sparseness Constraint

Abstract Recognising protein complexes in protein interaction networks is crucial, but poses a major challenge due to the frequency of noisy interactions. These networks typically involve numerous protein complexes, with each protein generally only participating in a few complexes. Current recognition models often ignore this aspect. To address this problem, we present a semi-supervised protein complex identification algorithm that extends non-negative matrix factorization (NMF) with sparsity constraints. In contrast to conventional approaches that apply a global sparsity constraint to the entire matrix, our method imposes individual sparsity constraints on protein membership indicator vectors. This targeted strategy controls the algorithm more effectively. Our experimental results with yeast and human protein interaction networks show that our algorithm achieves higher accuracy in identifying protein complexes than leading contemporary methods.

Regulation of tapioca starch 3D printability by yeast protein: Rheological, textural evaluation, and machine learning prediction

This article investigated the effects of yeast protein (YP) on gel rheology, texture, and 3D printability of tapioca starch and the feasibility of Principal component analysis (PCA) and support vector machine (SVM) algorithms for classification and prediction of printability. The results indicated that increasing YP content enhanced the viscosity, storage and loss moduli, and hardness, thereby improving extrudability and supportability of 3D printing. The addition of 15% YP exhibited the best 3D printing performance, but excessively high YP addition hindered ink extrusion. PCA analysis based on rheological and texture indices categorized the ink's 3D printing performance into four classes: poor support and low printing accuracy; good support but low printing accuracy; good support and high printing accuracy; and non-smooth extrusion. Furthermore, SVM algorithm used texture data to predict printability classification, with the highest prediction accuracy (91.67%) achieved at polynomial kernel among four different kernel functions. These results confirm that YP can serve as a potential ink for 3D printing and underscore SVM's efficacy in predicting ink's 3D printing performance.

Yeast protein‐derived γ‐glutamyl peptides prepared by transpeptidation reaction exhibit a pronounced taste‐enhancing effect

AbstractA high‐salt diet can induce hypertension, so salt reduction can prevent hypertension. γ‐Glutamyl peptides (GGPs) have obvious taste‐enhancing effects, while their contents in natural foods are relatively low. Yeast protein rich in Glu/Gln is a good precursor for the preparation of GGPs. In this study, yeast protein‐derived GGPs were prepared through hydrolysis and transpeptidation reactions, followed by sensory evaluation and E‐tongue analysis. Peptide sequences were identified by LC−MS/MS and screened for molecular docking. The optimal reaction conditions were hydrolysis for 4 h, enzyme concentration of 16 U/g, and transpeptidation for 4 h. GGPs could increase salt and umami intensity by 60.78% and 40.93% based on sensory evaluation, 22.52%, and 16.40% according to E‐tongue analysis. Fifteen γ‐glutamyl peptides with different peptide lengths were selected for molecular docking. Molecular docking confirmed their binding to calcium‐sensing receptors (CaSr) through hydrogen bonds and hydrophobic interaction, while interaction between CaSR receptor and γ‐glutamyl di‐, tri‐, and oligo‐peptides varied in binding energy. The stimulation received by CaSR lasted a longer time and varied in intensity. It was further proved that the flavor of mixed peptides has a layered sense and can give people a rich taste experience. Overall, yeast protein‐derived GGPs can enhance salt and umami taste, which can reduce salt usage without compromising taste.