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The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract Background The budding yeast Komagataella phaffii (Pichia pastoris) is widely employed to secrete proteins of academic and industrial interest. For secretory proteins, signal peptides are the sorting signal to direct proteins from cytosol to extracellular matrix, and their secretion efficiency directly impacts the yields of the targeted proteins in fermentation broth. Although the α-mating factor (MF) secretion signal from S. cerevisiae, the most common and widely used signal sequence for protein secretion, works in most cases, limitation exists as some proteins cannot be secreted efficiently. As the optimal choice of secretion signals is often protein specific, more secretion signals need to be developed to augment protein expression levels in K. phaffii. Results In this study, the secretion efficiency of 40 α-MF secretion signals from various yeast species and 32 endogenous signal peptides from K. phaffii were investigated using enhanced green fluorescent protein (EGFP) as the model protein. All of the evaluated α-MF secretion signals successfully directed EGFP secretion except for the secretion signals of the yeast D. hansenii CBS767 and H. opuntiae. The secretion efficiency of α-MF secretion signal from Wickerhamomyces ciferrii was higher than that from S. cerevisiae. 24 out of 32 endogenous signal peptides successfully mediated EGFP secretion. The signal peptides of chr3_1145 and FragB_0048 had similar efficiency to S. cerevisiae α-MF secretion signal for EGFP secretion and expression. Conclusions The screened α-MF secretion signals and endogenous signal peptides in this study confer an abundance of signal peptide selection for efficient secretion and expression of heterologous proteins in K. phaffii.

Using radiofrequency dielectric spectroscopy, we have investigated the impact of the interaction between a G protein-coupled receptor (GPCR), the sterile2 α-factor receptor protein (Ste2), and its cognate agonist ligand, the α-factor pheromone, on the dielectric properties of the plasma membrane in living yeast cells ( Saccharomyces cerevisiae ). The dielectric properties of a cell suspension containing a saturating concentration of α-factor were measured over the frequency range 40Hz-110 MHz and compared to the behavior of a similarly prepared suspension of cells in the absence of α-factor. A spherical three-shell model was used to determine the electrical phase parameters for the yeast cells in both types of suspensions. The relative permittivity of the plasma membrane showed a significant increase after exposure to α-factor (by 0.06 ± 0.05). The equivalent experiment performed on yeast cells lacking the ability to express Ste2 showed no change in plasma membrane permittivity. Interestingly, a large change also occurred to the electrical properties of the cellular interior after the addition of α-factor to the cell suspending medium, whether or not the cells were expressing Ste2. We present a number of different complementary experiments performed on the yeast to support these dielectric data and interpret the results in terms of specific cellular reactions to the presence of α-factor.

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Kex2 is a Ca 2+ -dependent serine protease from S. cerevisiae. Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S' of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR↓EADQ-EDDnp and Abz-YKR↓EADQ-EDDnp based on the pro-α-mating factor sequence. The specificity of Kex2 due to basic residues at P 1 ' is affected by the type of residue in the P 3 position. Some residues in P 1 ' with large or bulky side chains yielded poor substrate specificity. The k cat /K M values for peptides with P 2 ' substitutions containing Tyr in P 3 were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in k cat and K M , respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a polypeptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Abstract Background The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. Results Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57–70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30–43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47–49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30–43 in the WT-GCSF was partially restored (by 60%) when the Δ57–70 was added. The effect of Δ47–49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57–70 indicating the importance of the 47–49 region. A stimulatory effect of Δ57–70 was confirmed in the double deletion (Matα:Δ57–70;47–49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57–70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. Conclusions Among the different truncations (Matα:Δ57–70, Matα:Δ47–49, Matα:Δ30–43, Matα:Δ57–70;30–43, Matα:Δ57–70;47–49) in pro-peptide of α-MAT secretion signal, Matα:Δ57–70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30–43 and Matα:Δ47–49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

Background: The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production., Results: Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57-70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30-43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47-49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30-43 in the WT-GCSF was partially restored (by 60%) when the Δ57-70 was added. The effect of Δ47-49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57-70 indicating the importance of the 47-49 region. A stimulatory effect of Δ57-70 was confirmed in the double deletion (Matα:Δ57-70;47-49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57-70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system., Conclusions: Among the different truncations (Matα:Δ57-70, Matα:Δ47-49, Matα:Δ30-43, Matα:Δ57-70;30-43, Matα:Δ57-70;47-49) in pro-peptide of α-MAT secretion signal, Matα:Δ57-70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30-43 and Matα:Δ47-49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

The human granulocyte colony-stimulating factor (G-CSF) is one of the hematopoietic growth factors administered for chemotherapy induced neutropenia and is currently produced through recombinant route in Escherichia coli. The methylotrophic unicellular yeast Pichia pastoris (syn. Komagataella phaffii) makes a good host for production of human therapeutics as the proteins are low-mannose glycosylated, disulfide bonded and correctly folded on their way to the cell exterior. Given the low level of production of G-CSF in P. pastoris, the present study examined modification of the Saccharomyces cerevisiae derived α-mating type secretory signal sequence to enhance its production. The substitution of Glu, at the P1' position of the Kex2 cleavage site, by Val/Ala led to extracellular production of ~ 60 mg/L of G-CSF in the extracellular medium. Production was further increased to ~ 100 mg/L by putting these mutations against rarely occurring methanol slow utilization P. pastoris X-33 host. Analysis of the modelled structure of the signal peptide indicated exposed loop structures, created by presence of Val/Ala, that favour cleavage by the Kex2 peptidase thereby leading to enhanced production of G-CSF. The conformational changes, induced on account of binding between the signal sequence and the cargo protein (G-CSF), also appear to play an important role in the final yield of the extracellular protein., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The Saccharomyces cerevisiae α-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of STE2 identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer. Recent publication of a series of high-resolution cryo-EM structures of Ste2p in ligand-free, agonist-bound and antagonist-bound states now makes it possible to evaluate the results of these genetic and biochemical strategies, in comparison to three-dimensional structures showing activation-related conformational changes. The results indicate that the genetic and biochemical strategies were generally effective, and provide guidance as to how best to apply these experimental strategies to other proteins. These strategies continue to be useful in defining mechanisms of signal transduction in the context of the available structures and suggest aspects of receptor function beyond what can be discerned from the available structures.

Raw starch degrading enzyme specially glucoamylase with starch binding domain (SBD) has great values in the starch processing industry because it digests the starch particles below the gelatinization temperature by releasing glucose from the non-reducing ends sequentially. The purpose of the study was to measure the secretion levels of recombinant glucoamylase from Pichia pastoris, by using the a-mating factor secretion signal peptide (a-MF) and the native signal peptide of glucoamylase from Aspergillus flavus NSH9. The Aspergillus flavus NSH9 gene (with and without native signal sequences), encoding a pH and thermostable glucoamylase with an SBD, was successfully cloned and expressed in Pichia pastoris to produce recombinant glucoamylases. The constructed recombinant plasmids pPICZB_GA2 (having a native signal peptides) and pPICZaC_GA2 (having the a-MF) were 5144 and 5356 bp in length respectively. Recombinant pichia having a-MF signal sequence (plasmid, pPICZaC_GA2) gave the highest level of secretions of recombinant glucoamylase after 6 days of incubation period with 0.5% methanol. In conclusion, yeast expression vector signal peptide is more efficient for heterologous expression/secretions of recombinant glucoamylase compared to its native signal sequences. [ABSTRACT FROM AUTHOR]

The heterothallic group of the plant pathogen Phytophthora can sexually reproduce between the cross-compatible mating types A1 and A2. The mating hormone α2, produced by A2 mating type and utilized to promote the sexual reproduction of the partner A1 type, is known to be biosynthesized from phytol. In this study, we identified 2 biosynthetic intermediates, 11- and 16-hydroxyphytols (1 and 2), for α2 by administering the synthetic intermediates to an A2-type strain to produce α2 and by administering phytol to A2 strains to detect the intermediates in the mycelia. The results suggest that α2 is biosynthesized by possibly 2 cytochrome P450 oxygenases via 2 hydroxyphytol intermediates (1 and 2) in A2 hyphae and secreted outside., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating., Competing Interests: The authors have declared that no competing interests exist.

Haploid cells of the budding yeast Saccharomyces cerevisiae communicate using secreted pheromones and mate to form diploid zygotes. Mating is monogamous, resulting in the fusion of precisely one cell of each mating type. Monogamous mating in crowded conditions, where cells have access to more than one potential partner, raises the question of how multiple-mating outcomes are prevented. Here we identify mutants capable of mating with multiple partners, revealing the mechanisms that ensure monogamous mating. Before fusion, cells develop polarity foci oriented toward potential partners. Competition between these polarity foci within each cell leads to disassembly of all but one focus, thus favoring a single fusion event. Fusion promotes the formation of heterodimeric complexes between subunits that are uniquely expressed in each mating type. One complex shuts off haploid-specific gene expression, and the other shuts off the ability to respond to pheromone. Zygotes able to form either complex remain monogamous, but zygotes lacking both can re-mate.

Saccharomyces cerevisiae plays an important role in the heterologous expression of an array of proteins due to its easy manipulation, low requirements and ability for protein post-translational modifications. The implementation of the preproleader secretion signal of the α-factor mating pheromone from this yeast contributes to increase the production yields by targeting the foreign protein to the extracellular environment. The use of this signal peptide combined with enzyme-directed evolution allowed us to achieve the otherwise difficult functional expression of fungal laccases in S. cerevisiae, obtaining different evolved α-factor preproleader sequences that enhance laccase secretion. However, the design of a universal signal peptide to enhance the production of heterologous proteins in S. cerevisiae is a pending challenge. We describe here the optimisation of the α-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strategies: a bottom-up design over the native α-factor preproleader (α nat ) and a top-down design over the fittest evolved signal peptide obtained in our lab (α 9H2 leader). The goal was to analyse the effect of mutations accumulated in the signal sequence throughout iterations of directed evolution, or of other reported mutations, and their possible epistatic interactions. Both approaches agreed in the positive synergism of four mutations (Aα9D, Aα20T, Lα42S, Dα83E) contained in the final optimised leader (α OPT ), which notably enhanced the secretion of several fungal oxidoreductases and hydrolases. Additionally, we suggest a guideline to further drive the heterologous production of a particular enzyme based on combinatorial saturation mutagenesis of positions 86th and 87th of the α OPT leader fused to the target protein.

Twenty analogs of [Orn 6 ,D-Ala 9 ]α-factor were synthesized and assayed for their biological activities: seven analogs of [Orn 6 ,X 9 ]α-factor, seven analogs of [X 6 ,D-Ala 9 ]α-factor, five analogs of [X 5 ,X 6 ,D-Ala 9 ]α-factor, and native α-factor (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native α-factor (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU- STE2 vector. [Dap 6 ,D-Ala 9 ]α-factor with weak halo activity (10%) showed the highest receptor affinity (＞ 230%) and the highest gene induction activity (167%). [Arg 6 ,D-Ala 9 ]α-factor showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newly-designed fluorescence-based detector, [Arg 6 ,D-Ala 9 ]α-factor-Edan, with high sensitivity (12,500-fold higher than the absorption-based detector [Orn 6 ]α-factor-[Cys] 3 ).

G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast G-alpha (Gpa1p) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fluorophore. Constitutive and position-dependent BRET signal was observed in the absence of agonist (α-factor). Upon the activation of the receptors with α-factor, no significant change in BRET signal was observed. The location of Ste2p-Gpa1p heterodimer was investigated using confocal fluorescence microscopy and bimolecular fluorescence complementation (BiFC) assay, a technique where two non-fluorescent fragments of a fluorescent protein reassemble in vivo to restore fluorescence property thereby directly reporting a protein-protein interaction. BiFC experiments resulted in a dimerization signal intracellularly during biosynthesis on the endoplasmic reticulum (ER) and on the plasma membrane (PM). The constitutive BRET and BiFC signals observed on ER between Ste2p and Gpa1p in their quiescent and activated states are indicative of pre-coupling between these two proteins. This study is the first to show that the extreme N-terminus of yeast G protein alpha subunit is in close proximity to its receptor. The data suggests a pre-coupled heterodimer prior to receptor activation. The images presented in this study are the first direct in vivo evidence showing the localization of receptor - G protein heterodimers during biosynthesis and before reaching the plasma membrane., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. Hence, it plays a critical role in establishing many major human ailments, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dodecameric peptide that undergoes prenylation and subsequent processing in a manner identical to larger proteins. Due to its small size in addition to its well-characterized behavior in yeast, a-factor is an attractive model system to study the prenylation pathway. Traditionally, chemical synthesis and characterization of a-factor have been challenging, which has limited its use in prenylation studies. In this chapter, a robust method for the synthesis of a-factor is presented along with a description of the characterization of the peptide using MALDI and NMR. Finally, complete assignments of resonances from the isoprenoid moiety and a-factor from COSY, TOCSY, HSQC, and long-range HMBC NMR spectra are presented. This methodology should be useful for the synthesis and characterization of other mature prenylated peptides and proteins., (© 2019 Elsevier Inc. All rights reserved.)

In the human pathogenic mold Aspergillus fumigatus, sexual identity is determined by the mating-type idiomorphs MAT1-1 and MAT1-2 residing at the MAT locus. Upon crossing of compatible partners, a heterothallic mating is executed to eventually form cleistothecia that contain recombinant ascospores. Given that the MAT1 gene products are DNA binding master regulators that govern this complex developmental process, we monitored the MAT1-driven transcriptomes of A. fumigatus by conditional overexpression of either MAT1 gene followed by RNA-seq analyses. Numerous genes related to the process of mating were found to be under transcriptional control, such as pheromone production and recognition. Substantial differences between the MAT1-1- and MAT1-2-driven transcriptomes could be detected by functional categorization of differentially expressed genes. Moreover, a significant and distinct impact on expression of genetic clusters of secondary metabolism became apparent, which could be verified on the product level. Unexpectedly, specific cross-regulation of the fumagillin/pseurotin supercluster was evident, thereby uncoupling its co-regulatory characteristic. These insights imply a tight interconnection of sexual development accompanied by ascosporogenesis with secondary metabolite production of a pathogenic fungus and impose evolutionary constraints that link these two fundamental aspects of the fungal lifestyle., (© 2018 John Wiley & Sons Ltd.)

The mating of fungi depends on pheromones that mediate communication between two mating types. Most species use short peptides as pheromones, which are either unmodified (e.g., α-factor in Saccharomyces cerevisiae) or C-terminally farnesylated (e.g., a-factor in S. cerevisiae). Peptide pheromones have been found by genetics or biochemistry in a small number of fungi, but their short sequences and modest conservation make it impossible to detect homologous sequences in most species. To overcome this problem, we used a four-step computational pipeline to identify candidate a-factor genes in sequenced genomes of the Saccharomycotina, the fungal clade that contains most of the yeasts: we require that candidate genes have a C-terminal prenylation motif, are shorter than 100 amino acids long, and contain a proteolytic-processing motif upstream of the potential mature pheromone sequence and that closely related species contain highly conserved homologs of the potential mature pheromone sequence. Additional manual curation exploits the observation that many species carry more than one a-factor gene, encoding identical or nearly identical pheromones. From 332 Saccharomycotina genomes, we identified strong candidate pheromone genes in 241 genomes, covering 13 clades that are each separated from each other by at least 100 million years, the time required for evolution to remove detectable sequence homology among small pheromone genes. For one small clade, the Yarrowia, we demonstrated that our algorithm found the a-factor genes: deleting all four related genes in the a-mating type of Yarrowia lipolytica prevents mating., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The structures of seven synthetic transmembrane domains (TMDs) of the alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae were studied in phospholipid multilayers by transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies. Peptide conformation assumed in multilayers depended on the method of sample preparation. Amide proton H/D exchange experiments showed that 60-80% of the NH bonds in these TMDs did not exchange with bulk water in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilayers. FTIR results showed that peptides corresponding to TMDs one, two, and seven were mostly alpha-helical in DMPC multilayers. Peptides corresponding to TMDs three and six assumed predominantly beta-sheet structures, whereas those corresponding to TMDs four and five were a mixture of alpha-helices and beta-sheets. ATR-FTIR showed that in DMPC the alpha-helices of TMDs two and five oriented with tilt angles of 34 degrees and 32 degrees, respectively, with respect to the multilayer normal. Similar results were obtained for six of the transmembrane domains in DMPC/DMPG (4:1) multilayers. In a mixture [POPC/POPE/POPS/PI/ergosterol (30:20:5:20:25)] which mimicked the lipid composition of the S. cerevisiae cell membrane, the percentage of alpha-helical structures found for TMDs one and five increased compared to those in DMPC and DMPC/DMPG (4:1) multilayers, and TMD six exhibited a mixture of beta-sheet ( approximately 60%) and alpha-helical ( approximately 40%) structure. These experiments provide biophysical evidence that peptides representing the seven transmembrane domains in Ste2p assume different structures and tilt angles within a membrane multilayer.

Three analogues of the alpha-mating factor pheromone of Saccharomyces cerevisiae containing the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group were synthesized that had high binding affinity to the receptor and retained biological activity. The fluorescence emission maximum of the NBD group in [K7(NBD),Nle(12)]-alpha-factor was blue shifted by 35 nm compared to buffer when the pheromone bound to its receptor. Fluorescence quenching experiments revealed that the NBD group in [K7(NBD),Nle(12)]-alpha-factor bound to the receptor was shielded from collision with iodide anion when in aqueous buffer. In contrast, the emission maximum of NBD in [K7(ahNBD),Nle(12)]-alpha-factor or [Orn7(NBD),Nle(12)]-alpha-factor was not significantly shifted and iodide anion efficiently quenched the fluorescence of these derivatives when they were bound to receptor. The fluorescence investigation suggests that when the alpha-factor is bound to its receptor, K7 resides in an environment that has both hydrophobic and hydrophilic groups within a few angstroms of each other.

In the fungus Ustilago maydis , sexual pheromones elicit mating resulting in an infective filament able to infect corn plants. Along this process a G2 cell cycle arrest is mandatory. Such as cell cycle arrest is initiated upon the pheromone recognition in each mating partner, and sustained once cell fusion occurred until the fungus enter the plant tissue. We describe that the initial cell cycle arrest resulted from inhibition of the nuclear transport of the mitotic inducer Cdc25 by targeting its importin, Kap123. Near cell fusion to take place, the increase on pheromone signaling promotes Cdc25 degradation, which seems to be important to ensure the maintenance of the G2 cell cycle arrest to lead the formation of the infective filament. This way, premating cell cycle arrest is linked to the subsequent steps required for establishment of the infection. Disabling this connection resulted in the inability of fungal cells to infect plants., Competing Interests: PB, SC, OV, GB, JP No competing interests declared, (© 2019, Bardetti et al.)

The mating-specific yeast Gα controls pheromone signaling by sequestering Gβγ and by regulating the Fus3 MAP kinase. Disrupting Gα-Fus3 interaction leads to severe defects in chemotropism. Because Gα concentrates at the chemotropic growth site where Fus3 is required for the phosphorylation of two known targets, we screened for additional proteins whose phosphorylation depends on pheromone stimulation and Gα-Fus3 interaction. Using a mutant form of Gα severely defective in Fus3-binding, Gα DSD , and quantitative mass spectrometry, fourteen proteins were identified as potential targets of Gα-recruited Fus3, ten of which were previously implicated in cell polarity and morphogenesis. To explore the biological relevance of these findings, we focused on the Spa2 polarisome protein, which was hypophosphorylated on multiple serine residues in pheromone-treated Gα DSD cells. Six sites were mutagenized to create the Spa2 6XSA mutant protein. Spa2 6XSA exhibited increased affinity for Fus3, consistent with a kinase-substrate interaction, and Spa2 6XSA cells exhibited dramatic defects in gradient sensing and zygote formation. These results suggest that Gα promotes the phosphorylation of Spa2 by Fus3 at the cortex of pheromone-stimulated cells, and that this mechanism plays a role in chemotropism. How the Gα-Fus3 signaling hub affects the other putative targets identified here has yet to be determined. SIGNIFICANCE: Previously, interaction between the G alpha protein, Gpa1, and the MAPK of the pheromone response pathway, Fus3, was shown to be important for efficient sensing of the pheromone gradient and for the maintenance of cell polarity during mating. Here we show that the underlying molecular mechanisms involve the phosphorylation of specific cortical targets of Gpa1/Fus3. These have been identified by quantitative phosphoproteomics using a mutant of Gpa1, which is defective in interacting with Fus3. One of these targets is the polarisome protein Spa2. Alanine substitution of the Spa2 phosphorylation sites targeted by Gpa1/Fus3 lead to a dramatic defect in pheromone gradient sensing and zygote formation. These results reveal how the G alpha protein and the MAPK control cell polarity in a prototypical model system. Our results have wider significance as similar mechanisms exist in higher eukaryotes and are involved in important biological such as neuron development, immunity, and cancer cell metastasis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

alpha-Factor, a 13-amino-acid pheromone secreted by haploid alpha cells of Saccharomyces cerevisiae, binds to Ste2p, a seven-transmembrane, G-protein-coupled receptor present on haploid alpha cells, to activate a signal transduction pathway required for conjugation and mating. To determine the structural requirements for alpha-factor activity, we developed a genetic screen to identify from random and semirandom libraries novel peptides that function as agonists or antagonists of Ste2p. The selection scheme was based on autocrine strains constructed to secrete random peptides and respond by growth to those that were either agonists or antagonists of Ste2p. Analysis of a number of peptides obtained by this selection procedure indicates that Trp1, Trp3, Pro8, and Gly9 are important for agonist activity specifically. His2, Leu4, Leu6, Pro10, a hydrophobic residue 12, and an aromatic residue 13 are important for both agonist and antagonist activity. Our results also show that activation of Ste2p can be achieved with novel, unanticipated combinations of amino acids. Finally, the results suggest the utility of this selection scheme for identifying novel ligands for mammalian G-protein-coupled receptors heterologously expressed in S. cerevisiae.

Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation.

Directed evolution of membrane receptors is challenging as the evolved receptor must not only accommodate a non-native ligand, but also maintain the ability to transduce the detection of the new ligand to any associated intracellular components. The G-protein coupled receptor (GPCR) superfamily is the largest group of membrane receptors. As members of the GPCR family detect a wide range of ligands, GPCRs are an incredibly useful starting point for directed evolution of user-defined analytical tools and diagnostics. The aim of this study was to determine if directed evolution of the yeast Ste2p GPCR, which natively detects the α-factor peptide, could yield a GPCR that detects Cystatin C, a human peptide biomarker. We demonstrate a generalizable approach for evolving Ste2p to detect peptide sequences. Because the target peptide differs significantly from α-factor, a single evolutionary step was infeasible. We turned to a substrate walking approach and evolved receptors for a series of chimeric intermediates with increasing similarity to the biomarker. We validate our previous model as a tool for designing optimal chimeric peptide steps. Finally, we demonstrate the clinical utility of yeast-based biosensors by showing specific activation by a C-terminally amidated Cystatin C peptide in commercially sourced human urine. To our knowledge, this is the first directed evolution of a peptide GPCR.

Extracellular protein production is primarily preferred to facilitate the downstream processes in recombinant protein production. Secretion of recombinant proteins is mediated by the processing of signal peptides in their N-terminal portion by the secretory mechanism of host expression systems. These molecular elements involved in secretion are functionally interchangeable between different species and secretion sequence screening is one of the widely used approaches to improve extracellular protein production. In this study, α-mating and protein internal repeats (PIR) secretion sequences isolated from different yeasts (Kluyveromyces lactis, Kluyveromyces marxianus and Hansenula polymorpha) were tested in Pichia pastoris for the production of two different proteins (α-amylase and xylanase) and compared with the well-known secretory signals, S. cerevisiae α-mating factor (Sc-MF) and P. pastoris protein internal repeats PIR (PpPIR). The results obtained showed the potential of signal sequences tested. Among the tested peptides, the highest yields were achieved with H. polymorpha protein internal repeats (HpPIR) and K. lactis α-mating factor (Kl-MF) for xylanase and K. marxianus protein internal repeats (KmPIR) and K. lactis α-mating factor (Kl-MF) for amylase. In further studies, these sequences can be evaluated as alternatives in the production of different proteins in P. pastoris and in the production of recombinant proteins in different expression systems., Competing Interests: Declaration of Competing Interest The author declares no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed. The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed. This produced the mature Pem-CMG peptide.

The process of cell fusion during mating of the yeast Saccharomyces cerevisiae is mediated by factors secreted by the mating partners. Spatial gradients of one of these mating factors, alpha-factor, polarized the growth of projections by MATa cells. The site of previous budding did not affect the direction of polarization, and subsequent budding was also polarized if mating factor was removed. Orientation occurred in the presence of nocodazole, suggesting that microtubules were not critical. At extremely low concentrations of alpha-factor, sst2-mutants (which in genetic studies do not discriminate between partners producing different amounts of alpha-factor) were able to polarize their projections. The sensitivity of this spatial sensing mechanism in wild-type cells is such that differences in receptor occupancy estimated to be about 1% are sufficient for orientation.