Transfer RNAs (tRNAs) serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. In addition to this canonical role, tRNAs are involved in other processes such as programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, with the help of Saccharomyces cerevisiae and Trypanosoma brucei, we demonstrate that those tRNAs that promote efficient SC-RT establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determine the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open an unexplored direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

• MeSH
• antikodon metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• proteosyntéza * MeSH
• ribozomální proteiny metabolismus   MeSH
• ribozomy * metabolismus   MeSH
• RNA transferová * metabolismus   genetika   chemie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terminační kodon * genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antikodon MeSH
• ribozomální proteiny MeSH
• RNA transferová * MeSH
• terminační kodon * MeSH

The Homo sapiens Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ in exchange for protons across cell membranes, and its dysfunction results in various pathologies. The activity of HsNHA2 is specifically inhibited by the flavonoid phloretin. Using bioinformatic modeling, we predicted two amino acids (R177 and S178) as being important for the binding of phloretin to the HsNHA2 molecule. Functional expression of HsNHA2 in Saccharomyces cerevisiae and its site-directed mutagenesis revealed that while the R177T mutation resulted in an antiporter that was less sensitive to phloretin, the S178T mutation enhanced the inhibitory effect of phloretin on HsNHA2. Our data corroborate the transport properties of HsNHA2 and its interactions with an inhibitor and can be helpful for the development of new therapeutics targeting this antiporter and its pleiotropic physiological functions.

• Klíčová slova
• Na+/H+ antiporter, human NHA2, phloretin inhibition, yeast,
• MeSH
• arginin metabolismus   MeSH
• floretin * farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace MeSH
• mutageneze cílená MeSH
• Na(+)-H(+) antiport metabolismus   genetika   antagonisté a inhibitory   chemie   MeSH
• Saccharomyces cerevisiae * metabolismus   genetika   účinky léků   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• floretin * MeSH
• Na(+)-H(+) antiport MeSH

The 5'-3' exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by Rai1, which is recruited to the termination site by Rtt103, an adaptor protein binding to the RNAPII C-terminal domain (CTD). The overall architecture of the Xrn2/Rat1-Rai1-Rtt103 complex remains unknown. We combined structural biology methods to characterize the torpedo complex from Saccharomyces cerevisiae and Chaetomium thermophilum. Comparison of the structures from these organisms revealed a conserved protein core fold of the subunits, but significant variability in their interaction interfaces. We found that in the mesophile, Rtt103 utilizes an unstructured region to augment a Rai1 β-sheet, while in the thermophile Rtt103 binds to a C-terminal helix of Rai1 via its CTD-interacting domain with an α-helical fold. These different torpedo complex assemblies reflect adaptations to the environment and impact complex recruitment to RNAPII.

• Klíčová slova
• NMR, RNAPII, cryo-EM, exonuclease, structure, termination, thermophiles, torpedo complex,
• MeSH
• Chaetomium * metabolismus   chemie   MeSH
• exoribonukleasy * chemie   metabolismus   genetika   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• RNA-polymerasa II metabolismus   chemie   MeSH
• Saccharomyces cerevisiae - proteiny * chemie   metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• exoribonukleasy * MeSH
• RAT1 protein, S cerevisiae MeSH Prohlížeč
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny * MeSH

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.

• MeSH
• ADP-ribosylační faktory metabolismus   genetika   MeSH
• Archaea * genetika   metabolismus   MeSH
• archeální proteiny metabolismus   genetika   chemie   MeSH
• Eukaryota genetika   metabolismus   MeSH
• eukaryotické buňky metabolismus   MeSH
• fylogeneze * MeSH
• GTP-fosfohydrolasy metabolismus   genetika   chemie   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• organely * metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ADP-ribosylační faktory MeSH
• archeální proteiny MeSH
• GTP-fosfohydrolasy MeSH

Sterols perform essential structural and signalling functions in living organisms. Ergosterol contributes to the fluidity, permeability, microdomain formation and functionality of proteins in the yeast membrane. In our study, desmosterol was the most successful at compensating for the lack of ergosterol in Saccharomyces cerevisiae, besides stigmasterol and sitosterol. These three sterols supported cell growth without causing severe morphological defects, unlike cholesterol, 7-dehydrocholesterol, lathosterol, cholestanol or lanosterol. Together with ergosterol, they were also able to bring the plasma membrane potential of hem1Δ cells closer to the level of the wild type. In addition, desmosterol conferred even higher thermotolerance to yeast than ergosterol. Some sterols counteracted the antifungal toxicity of polyenes, azoles and terbinafine to hem1Δ cells. Plant sterols (stigmasterol, sitosterol) and desmosterol ensured the glucose-induced activation of H+-ATPase in hem1Δ cells analogously to ergosterol, whereas cholesterol and 7-dehydrocholesterol were less effective. Exogenous ergosterol, stigmasterol, sitosterol, desmosterol and cholesterol also improved the growth of Candida glabrata and Candida albicans in the presence of inhibitory concentration of fluconazole. The proper incorporation of exogenous sterols into the membrane with minimal adverse side effects on membrane functions was mainly influenced by the structure of the sterol acyl chain, and less by their ring structures.

• Klíčová slova
• Ergosterol, H(+)-ATPase, Multidrug resistance, Plasma membrane, Yeast, diS-C(3)(3) assay,
• MeSH
• antifungální látky * farmakologie   MeSH
• buněčná membrána * účinky léků   metabolismus   fyziologie   MeSH
• desmosterol metabolismus   farmakologie   MeSH
• ergosterol * metabolismus   farmakologie   MeSH
• fungální léková rezistence * MeSH
• mikrobiální testy citlivosti MeSH
• protonové ATPasy * metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * účinky léků   enzymologie   fyziologie   metabolismus   MeSH
• sitosteroly metabolismus   farmakologie   MeSH
• steroly * metabolismus   farmakologie   MeSH
• stigmasterol metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antifungální látky * MeSH
• desmosterol MeSH
• ergosterol * MeSH
• protonové ATPasy * MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• sitosteroly MeSH
• steroly * MeSH
• stigmasterol MeSH

Mitochondrial morphology is an important parameter of cellular fitness. Although many approaches are available for assessing mitochondrial morphology in mammalian cells, only a few technically demanding and laborious methods are available for yeast cells. A robust, fully automated and user-friendly approach that would allow (1) segmentation of tubular and spherical mitochondria in the yeast Saccharomyces cerevisiae from conventional wide-field fluorescence images and (2) quantitative assessment of mitochondrial morphology is lacking. To address this, we compared Global thresholding segmentation with deep learning MitoSegNet segmentation, which we retrained on yeast cells. The deep learning model outperformed the Global thresholding segmentation. We applied it to segment mitochondria in strain lacking the MMI1/TMA19 gene encoding an ortholog of the human TCTP protein. Next, we performed a quantitative evaluation of segmented mitochondria by analyses available in ImageJ/Fiji and by MitoA analysis available in the MitoSegNet toolbox. By monitoring a wide range of morphological parameters, we described a novel mitochondrial phenotype of the mmi1Δ strain after its exposure to oxidative stress compared to that of the wild-type strain. The retrained deep learning model, all macros applied to run the analyses, as well as the detailed procedure are now available at https://github.com/LMCF-IMG/Morphology_Yeast_Mitochondria .

• Klíčová slova
• Deep learning, Mitochondria, Mmi1, Oxidative stress, TCTP, Yeast,
• MeSH
• deep learning MeSH
• fluorescenční mikroskopie metody   MeSH
• mitochondrie * metabolismus   MeSH
• oxidační stres MeSH
• počítačové zpracování obrazu * metody   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Saccharomyces cerevisiae - proteiny MeSH

The nuclear envelope (NE) separates translation and transcription and is the location of multiple functions, including chromatin organization and nucleocytoplasmic transport. The molecular basis for many of these functions have diverged between eukaryotic lineages. Trypanosoma brucei, a member of the early branching eukaryotic lineage Discoba, highlights many of these, including a distinct lamina and kinetochore composition. Here, we describe a cohort of proteins interacting with both the lamina and NPC, which we term lamina-associated proteins (LAPs). LAPs represent a diverse group of proteins, including two candidate NPC-anchoring pore membrane proteins (POMs) with architecture conserved with S. cerevisiae and H. sapiens, and additional peripheral components of the NPC. While many of the LAPs are Kinetoplastid specific, we also identified broadly conserved proteins, indicating an amalgam of divergence and conservation within the trypanosome NE proteome, highlighting the diversity of nuclear biology across the eukaryotes, increasing our understanding of eukaryotic and NPC evolution.

• Klíčová slova
• AlphaFold, Nucleus, comparative genomics, molecular evolution, nuclear lamina, nuclear pore complex,
• MeSH
• jaderný obal * metabolismus   MeSH
• jaderný pór metabolismus   MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• lidé MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• Trypanosoma * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplex proteinů jaderného póru MeSH

L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7 mM IPTG for 4 h at 30 °C) resulted in a significant amount of L-asparaginase with a titer of 93.52 μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production.

• Klíčová slova
• Escherichia coli, Saccharomyces cerevisiae, Cancer treatment, L-asparaginase, Recombinant production, Response surface methodology (RSM),
• MeSH
• asparaginasa * genetika   biosyntéza   metabolismus   MeSH
• Escherichia coli K12 genetika   enzymologie   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fermentace MeSH
• isopropylthiogalaktosid farmakologie   MeSH
• rekombinantní proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• asparaginasa * MeSH
• isopropylthiogalaktosid MeSH
• rekombinantní proteiny * MeSH

Homeostasis of cellular membranes is maintained by fine-tuning their lipid composition. Yeast lipid transporter Osh6, belonging to the oxysterol-binding protein-related proteins family, was found to participate in the transport of phosphatidylserine (PS). PS synthesized in the endoplasmic reticulum is delivered to the plasma membrane, where it is exchanged for phosphatidylinositol 4-phosphate (PI4P). PI4P provides the driving force for the directed PS transport against its concentration gradient. In this study, we employed an in vitro approach to reconstitute the transport process into the minimalistic system of large unilamellar vesicles to reveal its fundamental biophysical determinants. Our study draws a comprehensive portrait of the interplay between the structure and dynamics of Osh6, the carried cargo lipid, and the physical properties of the involved membranes, with particular attention to the presence of charged lipids and to membrane fluidity. Specifically, we address the role of the cargo lipid, which, by occupying the transporter, imposes changes in its dynamics and, consequently, predisposes the cargo to disembark in the correct target membrane.

• MeSH
• biologický transport MeSH
• buněčná membrána * metabolismus   MeSH
• fluidita membrány MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfatidylseriny metabolismus   MeSH
• proteiny vázající oxysterol MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• steroidní receptory metabolismus   MeSH
• unilamelární lipozómy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfatidylinositolfosfáty MeSH
• fosfatidylseriny MeSH
• phosphatidylinositol 4-phosphate MeSH Prohlížeč
• proteiny vázající oxysterol MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• steroidní receptory MeSH
• unilamelární lipozómy MeSH

Trk1 is the main K+ importer of Saccharomyces cerevisiae. Its proper functioning enables yeast cells to grow in environments with micromolar amounts of K+. Although the structure of Trk1 has not been experimentally determined, the transporter is predicted to be composed of four MPM (transmembrane segment - pore loop - transmembrane segment) motifs which are connected by intracellular loops. Of those, in particular the first loop (IL1) is unique in its length; it forms more than half of the entire protein. The deletion of the majority of IL1 does not abolish the transport activity of Trk1. However IL1 is thought to be involved in the modulation of the transporter's functioning. In this work, we prepared a series of internally shortened versions of Trk1 that lacked various parts of IL1, and we studied their properties in S. cerevisiae cells without chromosomal copies of TRK genes. Using this approach, we were able to determine that both N- and C-border regions of IL1 are necessary for the proper localization of Trk1. Moreover, the N-border part of IL1 is also important for the functioning of Trk1, as its absence resulted in a decrease in the transporter's substrate affinity. In addition, in the internal part of IL1, we newly identified a stretch of amino-acid residues that are indispensable for retaining the transporter's maximum velocity, and another region whose deletion affected the ability of Trk1 to adjust its affinity in response to external levels of K+.

• Klíčová slova
• Alkali-metal-cation homeostasis, First intracellular loop, K(+) importer, Regulation, Saccharomyces cerevisiae, Trk1,
• MeSH
• biologický transport MeSH
• draslík * metabolismus   MeSH
• proteiny přenášející kationty * metabolismus   genetika   chemie   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   chemie   MeSH
• Saccharomyces cerevisiae * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík * MeSH
• proteiny přenášející kationty * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč