Programmed translational bypassing is a process whereby ribosomes “ignore” a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this ...puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a “takeoff codon” immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching “landing triplet” 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions.
Ionophores such as valinomycin and nigericin are potent tools for studying the impact of ion perturbance on cellular functions. To obtain a broader picture about molecular components involved in ...mediating the effects of these drugs on yeast cells under respiratory growth conditions, we performed a screening of the haploid deletion mutant library covering the Saccharomyces cerevisiae nonessential genes. We identified nearly 130 genes whose absence leads either to resistance or to hypersensitivity to valinomycin and/or nigericin. The processes affected by their protein products range from mitochondrial functions through ribosome biogenesis and telomere maintenance to vacuolar biogenesis and stress response. Comparison of the results with independent screenings performed by our and other laboratories demonstrates that although mitochondria might represent the main target for both ionophores, cellular response to the drugs is very complex and involves an intricate network of proteins connecting mitochondria, vacuoles, and other membrane compartments.
Glycerophosphocholine is formed via the deacylation of the phospholipid phosphatidylcholine. The protein encoded by Saccharomyces cerevisiae open reading frame YPL110c effects glycerophosphocholine ...metabolism in vivo, most likely by acting as a glycerophosphocholine phosphodiesterase. Deletion of YPL110c causes an accumulation of glycerophosphocholine in cells prelabeled with 14Ccholine. Correspondingly, overexpression of YPL110c results in reduced intracellular glycerophosphocholine in cells prelabeled with 14Ccholine. Glycerophospho3Hcholine supplied in the growth medium accumulates to a much greater extent in the intracellular fraction of a YPL110Δ strain than in a wild type strain. Furthermore, glycerophospho3Hcholine accumulation requires the transporter encoded by GIT1, a known glycerophosphoinositol transporter. Growth on glycerophosphocholine as the sole phosphate source requires YPL110c and the Git1p permease. In contrast to glycerophosphocholine, glycerophosphoinositol metabolism is unaffected by deletion of YPL110c. The open reading frame YPL110c has been termed GDE1.
Sec14 family of lipid transfer proteins in yeasts Holič, Roman; Šťastný, Dominik; Griač, Peter
Biochimica et biophysica acta. Molecular and cell biology of lipids,
October 2021, 2021-10-00, 20211001, Letnik:
1866, Številka:
10
Journal Article
Recenzirano
The hydrophobicity of lipids prevents their free movement across the cytoplasm. To achieve highly heterogeneous and precisely regulated lipid distribution in different cellular membranes, lipids are ...transported by lipid transfer proteins (LTPs) in addition to their transport by vesicles. Sec14 family is one of the most extensively studied groups of LTPs. Here we provide an overview of Sec14 family of LTPs in the most studied yeast Saccharomyces cerevisiae as well as in other selected non-Saccharomyces yeasts—Schizosaccharomyces pombe, Kluyveromyces lactis, Candida albicans, Candida glabrata, Cryptococcus neoformans, and Yarrowia lipolytica. Discussed are specificities of Sec14-domain LTPs in various yeasts, their mode of action, subcellular localization, and physiological function. In addition, quite few Sec14 family LTPs are target of antifungal drugs, serve as modifiers of drug resistance or influence virulence of pathologic yeasts. Thus, they represent an important object of study from the perspective of human health.
The fission yeast Schizosaccharomyces pombe is an important model organism for the study of fundamental questions in eukaryotic cell and molecular biology. A plethora of cellular processes are ...membrane associated and/or dependent on the proper functioning of cellular membranes. Phospholipids are not only the basic building blocks of cellular membranes; they also serve as precursors to numerous signaling molecules. In this review, we describe the biosynthetic pathways leading to major S. pombe phospholipids, how these pathways are regulated, and what is known about degradation and turnover of fission yeast phospholipids. This review also addresses the synthesis, regulation and the role of water‐soluble phospholipid precursors. The last chapter of the review is devoted to the use of S. pombe for the biotechnological production of value‐added lipid molecules.
Lipids are hydrophobic molecules with several key functions in the cell. Due to the thermodynamic constraints placed upon lipids, they cannot freely diffuse across the cytosol. Their transport ...between two membrane compartments can be facilitated by lipid transfer proteins (LTPs). One of the most studied families of LTPs is S. cerevisiae’s Sec14p family. Proteins belonging to this group contain cellular retinaldehyde binding – triple functional (CRAL‐TRIO) domain. In S. cerevisiae there are six members of this family, the founding member Sec14p and five of its homologues termed Sfh (Sec fourteen homologue) 1‐5. The Sec14p has been extensively studied and it was determined that it possesses phosphatidylinositol (PI) and phosphatidylcholine (PC) binding and transfer activities (Bankaitis et al., 1990). Many of Sfh proteins have been implicated in various processes in cell, however, their exact roles remain to be discovered.
Lipid binding and transfer by Sfh proteins were intensively studied (Li et al., 2000; Maeda et al., 2013; Tripathi et al., 2019; Holic et al., 2014; Pevalova et al., 2019) but some key questions remain open. Our objective is to determine lipids bound to S. cerevisiae’s Sec fourteen homologue proteins. For this we used HL‐60 in situ lipid‐binding assay, which employs co‐incubation of permeabilized human HL‐60 cells with recombinant protein of interest. Afterwards, the proteins are re‐purified using affinity chromatography. The lipids extracted from re‐isolated proteins are separated using thin layer chromatography and identified.
Our results showed that Sfh1 binds PC and PI with approximately the same affinity. The remaining Sfh proteins, excluding Sfh5p, predominantly bind PI and neutral lipids (NLs). Sfh5 was found to bind only PI. Analysis of bound NLs identified squalene in Sfh2p as a likely binding partner and cholesterol in association with Sfh3p and Sfh4p. Moreover, it appears that both Sfh3p and Sfh4p can bind another NL in addition to cholesterol, which remains to be identified.
Based on obtained results at least three out of five Sfh proteins are capable to bind NLs, predominantly sterols or precursors of the sterol biosynthetic pathway. It appears that binding of NLs is very specific, suggesting their crucial role in the function of Sfh proteins. Thus, the activity of some members of LTP family might be regulated by sterols or their precursors which in turn can connect and synchronize the sterol biosynthetic pathway with the phospholipid trafficking events. To identify the role of sterol binding by Sfh proteins in the physiology of the yeast cell, we will attempt to prepare mutants in sterol binding. References: Bankaitis et al. (1990) Nature 347: 561Li et al. (2000) Mol Biol Cell 11: 1989Maeda et al. (2013) Nature 501: 257Tripathi et al. (2019) JBC 294:19081Holic et al. (2014) BBA 1842:1483Pevalova et al. (2019) BBA 1864: 1412
Yeast Sec14‐like phosphatidylinositol transfer proteins (PITPs) contain a hydrophobic cavity capable of accepting a single molecule of phosphatidylinositol (PI) or another molecule in a mutually ...exclusive manner. We report here that two yeast Sec14 family PITPs, Pdr16p (Sfh3p) and Pdr17p (Sfh4p), possess high‐affinity binding and transfer towards lanosterol. To our knowledge, this is the first identification of lanosterol transfer proteins. In addition, a pdr16Δpdr17Δ double mutant had a significantly increased level of cellular lanosterol compared with the corresponding wild‐type. Based on the lipid profiles of wild‐type and pdr16Δpdr17Δ cells grown in aerobic and anaerobic conditions, we suggest that PI‐lanosterol transfer proteins are important predominantly for the optimal functioning of the post‐lanosterol part of sterol biosynthesis.
Yeast Sec14 family phosphatidylinositol (PI) transfer proteins Pdr16p (Sfh3p) and Pdr17p (Sfh4p) possess high‐affinity binding and transfer towards lanosterol. In addition, a pdr16Δpdr17Δ double mutant had a significantly increased level of cellular lanosterol. Based on the lipid profiles of pdr16Δpdr17Δ cells grown in aerobic and anaerobic conditions, we suggest that PI‐lanosterol transfer proteins are important predominantly for the optimal functioning of the post‐lanosterol part of sterol biosynthesis.
Sec14 related proteins in yeast Griac, Peter
Biochimica et biophysica acta,
06/2007, Letnik:
1771, Številka:
6
Journal Article
Recenzirano
Lipid transport between membranes of eukaryotic organisms represents an essential aspect of organelle biogenesis. This transport must be strictly selective and directional to assure specific lipid ...composition of individual membranes. Despite the intensive research effort in the last few years, our understanding of how lipids are sorted and moved within cells is still rather limited. Evidence indicates that at least some of the mechanisms generating and maintaining non-random distribution of lipids in cells are linked to the action of phosphatidylinositol transfer proteins (PITPs). The major PITP in yeast
Saccharomyces cerevisiae, Sec14p, is essential in promoting Golgi secretory function by modulating of its membrane lipid composition. This review focuses on a group of five yeast proteins that share significant sequence homology with Sec14p. Based on this sequence identity, they were termed Sfh (
Sec
fourteen
homologue) proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism, phosphoinositide mediated signaling and membrane trafficking.
Abstract
Squalene is a valuable natural substance with several biotechnological applications. In the yeast Saccharomyces cerevisiae, it is produced in the isoprenoid pathway as the first precursor ...dedicated to ergosterol biosynthesis. The aim of this study was to explore the potential of squalene epoxidase encoded by the ERG1 gene as the target for manipulating squalene levels in yeast. Highest squalene levels (over 1000 μg squalene per 109 cells) were induced by specific point mutations in ERG1 gene that reduced activity of squalene epoxidase and caused hypersensitivity to terbinafine. This accumulation of squalene in erg1 mutants did not significantly disturb their growth. Treatment with squalene epoxidase inhibitor terbinafine revealed a limit in squalene accumulation at 700 μg squalene per 109 cells which was associated with pronounced growth defects. Inhibition of squalene epoxidase activity by anaerobiosis or heme deficiency resulted in relatively low squalene levels. These levels were significantly increased by ergosterol depletion in anaerobic cells which indicated feedback inhibition of squalene production by ergosterol. Accumulation of squalene in erg1 mutants and terbinafine-treated cells were associated with increased cellular content and aggregation of lipid droplets. Our results prove that targeted genetic manipulation of the ERG1 gene is a promising tool for increasing squalene production in yeast.
The paper is aimed at utilization of specific mutants with reduced squalene epoxidase activity for increasing production of squalene as a biotechnologically valuable substance in the yeast Saccharomyces cerevisiae.
Prs PRPP (phosphoribosyl pyrophosphate) synthetase catalyses the transfer of pyrophosphate from ATP to ribose 5-phosphate, thereby activating the pentose sugar for incorporation into purine and ...pyrimidine nucleotides. The Saccharomyces cerevisiae genome contains five genes, PRS1-PRS5, whose products display characteristic PRPP and bivalent-cation-binding sites of Prs polypeptides. Deletion of one or more of the five PRS genes has far-reaching and unexpected consequences, e.g. impaired cell integrity, temperature-sensitivity and sensitivity to VPA (valproic acid) and LiCl. CTP pools in prs1Delta and prs3Delta are reduced to 12 and 31% of the wild-type respectively, resulting in an imbalance in phospholipid metabolism which may have an impact on the intracellular inositol pool which is affected by the administration of either VPA or LiCl. Overexpression of CTP synthetase in prs1Delta prs3Delta strains partially reverses the VPA-sensitive phenotype. Yeast two-hybrid screening revealed that Prs3 and the yeast orthologue of GSK3 (glycogen synthase kinase 3), Rim11, a serine/threonine kinase involved in several signalling pathways, interact with each other. Furthermore, Prs5, an essential partner of Prs3, which also interacts with GSK3 contains three neighbouring phosphorylation sites, typical of GSK3 activation. These studies on yeast PRPP synthetases bring together and expand the current theories for the mood-stabilizing effects of VPA and LiCl in bipolar disorder.
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