Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.

Cloning, expression, purification and biochemical characterization of the recombinant chitinase enzyme encoded by CTS2 in the budding yeast.

Chitin is a polymer of ß-1,4-linked N-acetylglucosamine (GlcNAc) and plays a central role in the assembly of the fungal cell wall. Chitinases are hydrolytic enzymes that break down glycosidic bonds in the chitin. Chitinases are classified into three categories, endochitinases, exochitinases and N-acetylglucosaminases, according to the manner in which the enzyme cleaves the chitin polymer. Saccharomyces cerevisiae has two chitinase-encoding genes, CTS1 and CTS2. However, whether Cts2p shows a chitinase activity remains unknown. In this study, we have cloned, expressed and purified the recombinant Cts2p protein from bacterial cells. We have demonstrated that Cts2p has a higher chitobiosidase (exochitinase) activity than endochitinase activity, but no N-acetylglucosaminase activity. The optimal temperature for the chitobiosidase activity of Cts2p is 37 °C.

Robust microtubule dynamics facilitate low-tension kinetochore detachment in metaphase.

During mitosis, sister chromatids are stretched apart at their centromeres via their attachment to oppositely oriented kinetochore microtubules. This stretching generates inwardly directed tension across the separated sister centromeres. The cell leverages this tension signal to detect and then correct potential errors in chromosome segregation, via a mechanical tension signaling pathway that detaches improperly attached kinetochores from their microtubules. However, the sequence of events leading up to these detachment events remains unknown. In this study, we used microfluidics to sustain and observe low-tension budding yeast metaphase spindles over multiple hours, allowing us to elucidate the tension history prior to a detachment event. We found that, under conditions in which kinetochore phosphorylation weakens low-tension kinetochore-microtubule connections, the mechanical forces produced via the dynamic growth and shortening of microtubules is required to efficiently facilitate detachment events. Our findings underscore the critical role of robust kinetochore microtubule dynamics in ensuring the fidelity of chromosome segregation during mitosis.

Human gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signalling in budding yeast.

Gasdermin D (GSDMD) and mixed lineage kinase domain-like protein (MLKL) are the pore-forming effectors of pyroptosis and necroptosis, respectively, with the capacity to disturb plasma membrane selective permeability and induce regulated cell death. The budding yeast Saccharomyces cerevisiae has long been used as a simple eukaryotic model for the study of proteins associated with human diseases by heterologous expression. In this work, we expressed in yeast both GSDMD and its N-terminal domain (GSDMD(NT)) to characterize their cellular effects and compare them to those of MLKL. GSDMD(NT) and MLKL inhibited yeast growth, formed cytoplasmic aggregates and fragmented mitochondria. Loss-of-function point mutants of GSDMD(NT) showed affinity for this organelle. Besides, GSDMD(NT) and MLKL caused an irreversible cell cycle arrest through TORC1 inhibition and disrupted endosomal and autophagic vesicular traffic. Our results provide a basis for a humanized yeast platform to study GSDMD and MLKL, a useful tool for structure-function assays and drug discovery.

Biological Activity of Optimized Codon Bovine Type III Interferon Expressed in Pichia pastoris.

Type III interferons (IFN-λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn-λ (boifn-λ) gene were synthetized after codon optimization. The boifn-λ gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN-λ3V18M. The recombinant plasmid pPICZαA-boIFN-λ3/λ3V18M was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high-level extracellular soluble form. Dominant expression strains of boIFN-λ3/λ3V18M were selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN-λ3/λ3V18M exceeded 106 U/mg, and they were neutralized with IFN-λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN-λ3/λ3V18M exerted antiproliferative effects on MDBK cells without cytotoxicity at 104 U/mL. Overall, boIFN-λ3 and boIFN-λ3V18M did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN-λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN-λs and provide material for therapeutic development.

Kodamaea samutsakhonensis f.a., sp. nov., a novel ascomycetous yeast species isolated from wild mushrooms in Thailand.

Two strains of genus Kodamaea, representing a novel anamorphic yeast species, were isolated from two samples of Marasmiellus sp. collected in Thailand. Analysis of the sequences of the internal transcribed spacer (ITS) regions and the D1/D2 domains of the large subunit (LSU) rRNA gene showed that the two strains differed by 27-42 nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 7-34 nucleotide substitutions in the ITS region of a group of related species, Kodamaea smagusa CBS 11430T, Kodamaea fungicola JCM 10142T, Kodamaea plutei ATCC MYA-4329T, Kodamaea lidongshanica SD5S01T and Kodamaea jinghongensis NYNU 167162T. Phylogenetic analysis based on the concatenated sequences of the ITS and the D1/D2 domains of the LSU rRNA gene showed that the two strains were placed in the Kodamaea clade and clearly separated from other recognized species of the genus. Therefore, the two strains were assigned as a novel species of the genus Kodamaea, for which we propose the name Kodamaea samutsakhonensis f.a., sp. nov. The holotype is TBRC 16043T (=DMKU-BP19T) and the isotype is PYCC 9354. The MycoBank number of the novel species is MB 846490.

Breaking spore dormancy in budding yeast transforms the cytoplasm and the solubility of the proteome.

The biophysical properties of the cytoplasm are major determinants of key cellular processes and adaptation. Many yeasts produce dormant spores that can withstand extreme conditions. We show that spores of Saccharomyces cerevisiae exhibit extraordinary biophysical properties, including a highly viscous and acidic cytosol. These conditions alter the solubility of more than 100 proteins such as metabolic enzymes that become more soluble as spores transit to active cell proliferation upon nutrient repletion. A key regulator of this transition is the heat shock protein, Hsp42, which shows transient solubilization and phosphorylation, and is essential for the transformation of the cytoplasm during germination. Germinating spores therefore return to growth through the dissolution of protein assemblies, orchestrated in part by Hsp42 activity. The modulation of spores' molecular properties are likely key adaptive features of their exceptional survival capacities.

The breaking of fungal spore dormancy: A coordinated transition.

The transition from dormant spore to germling has been topic of study and debate. A recent discovery in PLOS Biology shows that chaperone Hsp42 plays a crucial role in resolubilizing the proteome during dormancy breaking, although a role of trehalose cannot be excluded.

An inducible Komagataella phaffii system for protein expression using sorbitol dehydrogenase promoter.

OBJECTIVES: The aim of the present work was to develop a methanol-independent Komagataella phaffii (K. phaffii) strain using a non-methanol promoter. RESULTS: In this study, the food grade enzyme xylanase from Aspergillus niger ATCC 1015 was used as the reporter protein, a recombinant K. phaffii containing a cascade gene circus was designed and constructed using sorbitol as inducer. Sorbitol induced PSDH leading to MIT1 expression firstly, and heterologous protein xylanase expression finally. This system showed 1.7 fold of xylanase activity at the condition of single copy number of extra MIT1, and 2.1 fold of xylanase activity at condition of multi-copy extra MIT1 gene. CONCLUSIONS: This sorbitol-induced expression system of K. phaffii avoided toxic and explosive methanol. It was a novel cascade gene expression and a food safety system.

Mini-Bioreactor Platform for Membrane Protein Production in Komagataella pastoris.

Membrane proteins (MPs) play vital roles across various cellular functions, biological processes, physiological signaling pathways, and human-related disorders. Considering the clinical relevance of MPs and their application as therapeutic targets, it is crucial to explore highly effective production platforms and purification approaches to ultimately obtain a high-resolution structure of the target. Therefore, it would be possible to gather detailed knowledge on their mechanism of action which will be the basis for the rational design of novel and stronger drugs. Unfortunately, when compared to their soluble counterparts, 3D structures of MPs are really scarce (<2%), mainly due to poorly natural abundance, challenges associated with protein solubility and stability, and difficulties in producing bioactive and properly structural folded targets. These drawbacks could significantly impair the use of MPs as therapeutic targeting and demand efforts to develop tailor-made strategies for their appropriate handling. Therefore, this chapter is focused on describing a detailed and high-throughput procedure for the biosynthesis of MPs using Komagataella pastoris cell cultures as expression system in a mini-bioreactor platform. Additionally, insights on a purification strategy that combines immobilized-metal affinity and ion-exchange chromatography are described to further obtain the target protein with a significant degree of purity.

Optimal preparation of food waste to increase its utility for sophorolipid production by Starmerella bombicola.

Secondary feedstocks, such as food waste (FW), have been used for yeasts (e.g. Starmerella bombicola) to produce sophorolipids (SLs), which are commercially available biosurfactants. However, the quality of FW varies by location and season and may contains chemicals that inhibit SLs production. Therefore, it is crucial to identify such inhibitors and, if possible, remove them, to ensure efficient utilization. In this study, large scale FW was first analysed to determine the concentration of potential inhibitors. Lacticacid, acetic acid and ethanol were identified and found to be inhibitors of the growth of S. bombicola and its SLs production. Various methods were then evaluated for their ability to remove these inhibitors. Finally, a simple and effective strategy for removing inhibitors from FW was developed that complied with the 12 principles of green chemistry and could be adopted by industry for high SLs production.

Sugiyamaella bielyi f. a., sp. nov. and Sugiyamaella amazoniana f. a., sp. nov., two yeast species isolated from passalid beetles and rotting wood in Amazonia.

Sixteen yeast isolates representing two novel species of the genus Sugiyamaella were obtained from passalid beetles, their galleries and rotting wood collected in three sites of Amazonian Forest in Brazil. Sequence analyses of the ITS-5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the first species, described here as Sugiyamaella amazoniana f. a., sp. nov. (holotype CBS 18112, MycoBank 847461) is phylogenetically related to S. bonitensis with these species differing by 37 nucleotide substitutions and six gaps in D1/D2 sequences. S. amazoniana is represented by nine isolates obtained from the guts of the passalid beetles Popilius marginatus, Veturius magdalenae, Veturius sinuosus and Spasalus aquinoi, a beetle gallery and rotting wood. The second species, Sugiyamaella bielyi f. a., sp. nov. (holotype CBS 18148, MycoBank 847463), is most phylogenetically related to several undescribed Sugiyamaella species. S. bielyi is described based on seven isolates obtained from the guts of V. magdalenae and V. sinuosus, a beetle gallery and rotting wood. Both species appear to be associated with passalid beetles and their ecological niches in Amazonian biome.

Purification of human ß- and γ-actin from budding yeast.

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human ß- or γ-actin (i.e. cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both ß- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin and thymosin-ß4 (Tß4). Notably, Tß4 and profilin bind to ß- or γ-actin with higher affinity than to α-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies on actin regulation.

Oenological characteristics of two indigenous Starmerella bacillaris strains isolated from Chinese wine regions.

To broaden knowledge about the oenological characteristics of Starmerella bacillaris, the influence of two Chinese indigenous S. bacillaris strains on the conventional enological parameters and volatile compounds of Cabernet Sauvignon wines were investigated under different inoculation protocols (single inoculation and simultaneous/sequential inoculation with the commercial Saccharomyces cerevisiae EC1118). The results showed that the two S. bacillaris strains could complete alcohol fermentation alone under high sugar concentrations while increasing the content of glycerol and decreasing the content of acetic acid. Compared with wines fermented by EC1118 single inoculation, S. bacillaris single inoculation and S. bacillaris/EC1118 sequential inoculation increased the contents of isobutanol, ethyl isobutanoate, terpenes, and ketones and decreased the contents of isopentanol, phenylethyl alcohol, fatty acids, acetate esters, and total ethyl esters. Furthermore, for S. bacillaris/EC1118 simultaneous inoculation, the concentrations of ethyl esters were increased, contributing to a higher score of "floral" and "fruity" notes in agreement with sensory analysis. KEY POINTS: • S. bacillaris single and simultaneous/sequential inoculation. • Conventional enological parameters and volatile compounds were investigated. • S. bacillaris/EC1118 simultaneous fermentation increased ethyl esters.

Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of ß-Galactosidase in Pichia pastoris.

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of ß-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Nanozyme can substitute a natural Ogataea polymorpha catalase enzyme in vivo.

Nanomaterials possessing artificial, enzyme-like catalytic activity (nanozymes, NZs) have a great potential for application in research, immunological assays, biosensors, in vivo imaging, and as therapeutic agents. Despite the obvious advances in construction and understanding of functional properties of NZs, there is still no clear evidence of whether they can complement the loss of corresponding enzymatic activity in vivo. Herein, we report the first, to the best to our knowledge, example of successful substitution of natural enzyme activity by catalase-like platinum (nPt) and platinum-gold (nPtAu) nanoparticles transferred to the cells of methylotrophic yeast Ogataea polymorpha. The nPt NZs were synthesized by the chemical reduction method and used as a seed to produce the nPt(core)Au(shell) particles. The produced nPt NZs were 68.1 and 91.3 nm in size, while the hydrids were of 531.2 and 615.1 nm. Both nPt and nPtAu demonstrated catalase activity in vitro. The catalase-deficient strain Ogataea polymorpha C-105 was shown to be able to grow on methanol and a mixture of glucose and methanol in the presence although not in the absence of NZs, this correlating with the decrease in intracellular hydrogen peroxide production. The results provide the first example of complementation of the natural enzyme function by synthetic NZs, the phenomenon which can further be used in a screening for new catalase-like nanozymes and as a fruitful tool to modify living cells by nanoparticles possessing catalytic activity and to use such modified cells as sensitive elements in cell-based biosensors.

Transcriptomic profiling of the yeast Komagataella phaffii in response to environmental alkalinization.

BACKGROUND: Adaptation to alkalinization of the medium in fungi involves an extensive remodeling of gene expression. Komagataella phaffii is an ascomycetous yeast that has become an organism widely used for heterologous protein expression. We explore here the transcriptional impact of moderate alkalinization in this yeast, in search of suitable novel promoters able to drive transcription in response to the pH signal. RESULTS: In spite of a minor effect on growth, shifting the cultures from pH 5.5 to 8.0 or 8.2 provokes significant changes in the mRNA levels of over 700 genes. Functional categories such as arginine and methionine biosynthesis, non-reductive iron uptake and phosphate metabolism are enriched in induced genes, whereas many genes encoding iron-sulfur proteins or members of the respirasome were repressed. We also show that alkalinization is accompanied by oxidative stress and we propose this circumstance as a common trigger of a subset of the observed changes. PHO89, encoding a Na+/Pi cotransporter, appears among the most potently induced genes by high pH. We demonstrate that this response is mainly based on two calcineurin-dependent response elements located in its promoter, thus indicating that alkalinization triggers a calcium-mediated signal in K. phaffii. CONCLUSIONS: This work defines in K. phaffii a subset of genes and diverse cellular pathways that are altered in response to moderate alkalinization of the medium, thus setting the basis for developing novel pH-controlled systems for heterologous protein expression in this fungus.

Genetic Dissection of Budding Yeast PCNA Mutations Responsible for the Regulated Recruitment of Srs2 Helicase.

DNA-damage tolerance (DDT) is a mechanism by which eukaryotes bypass replication-blocking lesions to resume DNA synthesis and maintain cell viability. In Saccharomyces cerevisiae, DDT is mediated by sequential ubiquitination and sumoylation of proliferating cell nuclear antigen (PCNA, encoded by POL30) at the K164 residue. Deletion of RAD5 or RAD18, encoding two ubiquitin ligases required for PCNA ubiquitination, results in severe DNA-damage sensitivity, which can be rescued by inactivation of SRS2 encoding a DNA helicase that inhibits undesired homologous recombination. In this study, we isolated DNA-damage resistant mutants from rad5Δ cells and found that one of them contained a pol30-A171D mutation, which could rescue both rad5Δ and rad18Δ DNA-damage sensitivity in a srs2-dependent and PCNA sumoylation-independent manner. Pol30-A171D abolished physical interaction with Srs2 but not another PCNA-interacting protein Rad30; however, Pol30-A171 is not located in the PCNA-Srs2 interface. The PCNA-Srs2 structure was analyzed to design and create mutations in the complex interface, one of which, pol30-I128A, resulted in phenotypes reminiscent of pol30-A171D. This study allows us to conclude that, unlike other PCNA-binding proteins, Srs2 interacts with PCNA through a partially conserved motif, and the interaction can be strengthened by PCNA sumoylation, which turns Srs2 recruitment into a regulated process. IMPORTANCE It is known that budding yeast PCNA sumoylation serves as a ligand to recruit a DNA helicase Srs2 through its tandem receptor motifs that prevent unwanted homologous recombination (HR) at replication forks, a process known as salvage HR. This study reveals detailed molecular mechanisms, in which constitutive PCNA-PIP interaction has been adapted to a regulatory event. Since both PCNA and Srs2 are highly conserved in eukaryotes, from yeast to human, this study may shed light to investigation of similar regulatory mechanisms.

Systematic analysis of microtubule plus-end networks defines EB-cargo complexes critical for mitosis in budding yeast.

Microtubules are ubiquitous cytoskeletal polymers with essential functions in chromosome segregation, intracellular transport, and cellular morphogenesis. End-binding proteins (EBs) form the nodes of intricate microtubule plus-end interaction networks. Which EB binding partners are most critical for cell division and how cells organize a microtubule cytoskeleton in the absence of an EB protein are open questions. Here, we perform a detailed analysis of deletion and point mutants of the budding yeast EB protein Bim1. We demonstrate that Bim1 executes its key mitotic functions as part of two cargo complexes-Bim1-Kar9 in the cytoplasm and Bim1-Bik1-Cik1-Kar3 in the nucleus. The latter complex acts during initial metaphase spindle assembly and supports tension establishment and sister chromatid biorientation. We demonstrate that engineered plus-end targeting of Cik1-Kar3 and overexpression of the microtubule crosslinker Ase1 restore distinct aspects of the bim1Δ spindle phenotype. In addition to defining key Bim1-cargo complexes our study also characterizes redundant mechanisms that allow cells to proliferate in the absence of Bim1.