Erv14, a conserved cargo receptor of COPII vesicles, helps the proper trafficking of many but not all transporters to the yeast plasma membrane, for example, three out of five alkali-metal-cation transporters in Saccharomyces cerevisiae. Among them, the Nha1 cation/proton antiporter, which participates in cell cation and pH homeostasis, is a large membrane protein (985 aa) possessing a long hydrophilic C-terminus (552 aa) containing six conserved regions (C1-C6) with unknown function. A short Nha1 version, lacking almost the entire C-terminus, still binds to Erv14 but does not need it to be targeted to the plasma membrane. Comparing the localization and function of ScNha1 variants shortened at its C-terminus in cells with or without Erv14 reveals that only ScNha1 versions possessing the complete C5 region are dependent on Erv14. In addition, our broad evolutionary conservation analysis of fungal Na+ /H+ antiporters identified new conserved regions in their C-termini, and our experiments newly show C5 and other, so far unknown, regions of the C-terminus, to be involved in the functionality and substrate specificity of ScNha1. Taken together, our results reveal that also relatively small hydrophilic parts of some yeast membrane proteins underlie their need to interact with the Erv14 cargo receptor.

• MeSH
• antiportéry genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• COP-vezikuly genetika   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• membránové proteiny metabolismus   fyziologie   MeSH
• proteiny přenášející kationty metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sodík metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

• MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• geneticky modifikované organismy MeSH
• proteosyntéza genetika   MeSH
• ribozomální proteiny genetika   fyziologie   MeSH
• ribozomy metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   fyziologie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terminace translace peptidového řetězce * genetika   MeSH
• terminační kodon metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity.

• MeSH
• acetylace MeSH
• aktivace transkripce MeSH
• časové faktory MeSH
• chromatin metabolismus   MeSH
• dlouhověkost MeSH
• down regulace MeSH
• histonacetyltransferasy metabolismus   MeSH
• histony metabolismus   MeSH
• kalorická restrikce * MeSH
• N-terminální acetyltransferáza D nedostatek   genetika   fyziologie   MeSH
• nikotinamidasa genetika   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• regulace genové exprese u hub * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.

• MeSH
• "flap" endonukleasy fyziologie   MeSH
• DNA primery MeSH
• DNA vazebné proteiny fyziologie   MeSH
• DNA-helikasy fyziologie   MeSH
• endonukleasy fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• polymerázová řetězová reakce MeSH
• rekombinace genetická * MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence nukleotidů MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.

• MeSH
• DNA metabolismus   MeSH
• endonukleasy chemie   genetika   metabolismus   MeSH
• enzymy opravy DNA chemie   genetika   metabolismus   MeSH
• intracelulární signální peptidy a proteiny fyziologie   MeSH
• lysin metabolismus   MeSH
• mutace MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• protein-serin-threoninkinasy fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   fyziologie   MeSH
• sumoylace * MeSH
• ubikvitinligasy fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Cation-Cl(-) cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl(-) with Na(+) and/or K(+) ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K(+) uptake produced a significant growth advantage over the parental strain under K(+)-limiting conditions, and a hypersensitivity to the exogenous K(+)/H(+) exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K(+)-Cl(-) cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolar protein homologous to CCC family 1).

• MeSH
• chloridy chemie   MeSH
• DNA chemie   MeSH
• elektrochemie metody   MeSH
• fluorescenční mikroskopie metody   MeSH
• fylogeneze MeSH
• genotyp MeSH
• iontový transport MeSH
• kationty chemie   MeSH
• koncentrace vodíkových iontů MeSH
• konfokální mikroskopie metody   MeSH
• membránové potenciály MeSH
• nigericin farmakologie   MeSH
• oligonukleotidy chemie   MeSH
• osmotický tlak MeSH
• otevřené čtecí rámce MeSH
• rekombinace genetická MeSH
• Saccharomyces cerevisiae - proteiny chemie   fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sorbitol chemie   MeSH
• symportéry chemie   fyziologie   MeSH
• vakuoly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis.

• MeSH
• buněčná membrána fyziologie   MeSH
• endocytóza MeSH
• endoplazmatické retikulum fyziologie   MeSH
• fosfoproteiny fyziologie   MeSH
• klathrin fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.

• MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• genový knockout MeSH
• iniciace translace peptidového řetězce * MeSH
• interakční proteinové domény a motivy MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• podjednotky proteinů metabolismus   MeSH
• ribozomální proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Each of the three plasma membrane Ato proteins is involved in ammonium signalling and the development of yeast colonies. This suggests that although these proteins are homologous, they do not functionally substitute for each other, but may form a functional complex. Here, we present a detailed combined FRET, FLIM and photobleaching study, which enabled us to detect interactions between Ato proteins found in distinct compartments of yeast cells. We thus show that the proteins Ato1p and Ato2p interact and can form complexes when present in the plasma membrane. No interaction was detected between Ato1p and Ato3p or Ato2p and Ato3p. In addition, using specially prepared strains, we were able to detect an interaction between molecules of the same Ato protein, namely Ato1p-Ato1p and Ato3p-Ato3p, but not Ato2p-Ato2p.

• MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• fluorescenční spektrometrie metody   MeSH
• FRAP MeSH
• mapování interakce mezi proteiny MeSH
• membránové proteiny metabolismus   fyziologie   MeSH
• membránové transportní proteiny metabolismus   fyziologie   MeSH
• regulace genové exprese u hub MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae chemie   metabolismus   MeSH
• signální transdukce MeSH
• subcelulární frakce metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Saccharomyces cerevisiae general amino acid permease Gap1 (ScGap1) not only mediates the uptake of most amino acids but also functions as a receptor for the activation of protein kinase A (PKA). Fungal pathogens can colonize different niches in the host, each containing various levels of different amino acids and sugars. The Candida albicans genome contains six genes homologous to the S. cerevisiae GAP1. The expression of these six genes in S. cerevisiae showed that the products of all six C. albicans genes differ in their transport capacities. C. albicans Gap2 (CaGap2) is the true orthologue of ScGap1 as it transports all tested amino acids. The other CaGap proteins have narrower substrate specificities though CaGap1 and CaGap6 transport several structurally unrelated amino acids. CaGap1, CaGap2, and CaGap6 also function as sensors. Upon detecting some amino acids, e.g., methionine, they are involved in a rapid activation of trehalase, a downstream target of PKA. Our data show that CaGAP genes can be functionally expressed in S. cerevisiae and that CaGap permeases communicate to the intracellular signal transduction pathway similarly to ScGap1.

• MeSH
• Candida albicans genetika   metabolismus   MeSH
• geny hub MeSH
• molekulární sekvence - údaje MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• regulace genové exprese u hub genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence nukleotidů MeSH
• transportní systémy aminokyselin genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH