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Autophagy Oct 2023Macroautophagy/autophagy is a highly conserved pathway of cellular degradation and recycling that maintains cell health during homeostatic conditions and facilitates...
Macroautophagy/autophagy is a highly conserved pathway of cellular degradation and recycling that maintains cell health during homeostatic conditions and facilitates survival during stress. Aberrant cellular autophagy contributes to the pathogenesis of human diseases such as cancer, neurodegeneration, and cardiovascular, metabolic and lysosomal storage disorders. Despite decades of research, there remain unanswered questions as to how autophagy modulates cellular metabolism, and, conversely, how cellular metabolism affects autophagy activity. Here, we have identified the yeast metabolic transcription factor Stb5 as a negative regulator of autophagy. Chromosomal deletion of in the yeast enhances autophagy. Loss of Stb5 results in the upregulation of select uophay-related () transcripts under nutrient-replete conditions; however, the Stb5-mediated impact on autophagy occurs primarily through its effect on genes involved in NADPH production and the pentose phosphate pathway. This work provides insight into the intersection of Stb5 as a transcription factor that regulates both cellular metabolic responses and autophagy activity.: bp, base pairs; ChIP, chromatin immunoprecipitation; G6PD, glucose-6-phosphate dehydrogenase; GFP, green fluorescent protein; IDR, intrinsically disordered region; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); ORF, open reading frame; PA, protein A; PCR, polymerase chain reaction; PE, phosphatidylethanolamine; PPP, pentose phosphate pathway; prApe1, precursor aminopeptidase I; ROS, reactive oxygen species; RT-qPCR, real-time quantitative PCR; SD, standard deviation; TF, transcription factor; TOR, target of rapamycin; WT, wild-type.
Topics: Humans; Autophagy; Gene Expression Regulation, Fungal; NADP; Saccharomyces cerevisiae; Transcription Factors
PubMed: 37345792
DOI: 10.1080/15548627.2023.2228533 -
Molecular Cell Mar 2024The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the...
The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.
Topics: Cell Cycle Proteins; Saccharomyces cerevisiae Proteins; Chromosomal Proteins, Non-Histone; DNA, Superhelical; Cohesins; DNA; Saccharomyces cerevisiae; Chromosomes
PubMed: 38295804
DOI: 10.1016/j.molcel.2024.01.005 -
Cell Reports Nov 2023Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation,...
Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation, however, is unclear. We therefore perform Ribo-seq and Disome-seq in Saccharomyces cerevisiae and show that oxidative stress causes ribosome pausing at specific amino acid motifs, which also leads to ribosome collisions. However, these redox-pausing signatures are lost in the absence of Rad6 and do not depend on the ribosome-associated quality control (RQC) pathway. We also show that Rad6 is needed to inhibit overall translation in response to oxidative stress and that its deletion leads to increased expression of antioxidant genes. Finally, we observe that the lack of Rad6 leads to changes during translation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.
Topics: Ubiquitin; Saccharomyces cerevisiae Proteins; gamma-Glutamyl Hydrolase; Saccharomyces cerevisiae; Ribosomes; Oxidative Stress
PubMed: 37917585
DOI: 10.1016/j.celrep.2023.113359 -
Journal of Agricultural and Food... Jul 2023Linalool, a plant-derived high-value monoterpene, is widely used in the perfume, cosmetic, and pharmaceutical industries. Recently, engineering microbes to produce...
Linalool, a plant-derived high-value monoterpene, is widely used in the perfume, cosmetic, and pharmaceutical industries. Recently, engineering microbes to produce linalool has become an attractive alternative to plant extraction or chemical synthesis approaches. However, the low catalytic activity of linalool synthase and the shortage of precursor pools have been considered as two key factors for low yields of linalool. In this study, we rationally engineered the entrance of the substrate-binding pocket of linalool synthase (t67OMcLIS) and successfully increased the catalytic efficiency of this enzyme toward geranyl pyrophosphate. Specifically, F447E and F447A, with decreased entrance hydrophobicity and steric hindrance, increased linalool production by 2.2 and 1.9 folds, respectively. Subsequently, cytoplasm and peroxisomes were harnessed to boost linalool synthesis in , achieving a high titer of linalool (219.1 mg/L) in shake-flask cultivation. Finally, the engineered diploid strain produced 2.6 g/L of linalool by 5 L fed-batch fermentation, which was the highest production in yeast to date. The protein engineering and biosynthetic pathway compartmentalization in the peroxisome provide references for the microbial production of other monoterpenes.
Topics: Saccharomyces cerevisiae; Acyclic Monoterpenes; Monoterpenes; Proteins; Organelles; Metabolic Engineering
PubMed: 37350414
DOI: 10.1021/acs.jafc.2c08416 -
Methods in Cell Biology 2024Chronological age represents the time that passes between birth and a given date. To understand the complex network of factors contributing to chronological lifespan, a...
Chronological age represents the time that passes between birth and a given date. To understand the complex network of factors contributing to chronological lifespan, a variety of model organisms have been implemented. One of the best studied organisms is the yeast Saccharomyces cerevisiae, which has greatly contributed toward identifying conserved biological mechanisms that act on longevity. Here, we discuss high- und low-throughput protocols to monitor and characterize chronological lifespan and chronological aging-associated cell death in S. cerevisiae. Included are propidium iodide staining with the possibility to quantitatively assess aging-associated cell death via flow cytometry or qualitative assessments via microscopy, cell viability assessment through plating and cell counting and cell death characterization via propidium iodide/AnnexinV staining and subsequent flow cytometric analysis or microscopy. Importantly, all of these methods combined give a clear picture of the chronological lifespan under different conditions or genetic backgrounds and represent a starting point for pharmacological or genetic interventions.
Topics: Saccharomyces cerevisiae; Propidium; Saccharomyces cerevisiae Proteins
PubMed: 38302246
DOI: 10.1016/bs.mcb.2022.09.006 -
ACS Synthetic Biology May 2024Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details...
Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of . Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. This study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.
Topics: Saccharomyces cerevisiae; Metabolic Engineering; Models, Biological; Signal Transduction; Metabolic Networks and Pathways; Gene Expression Regulation, Fungal
PubMed: 38657170
DOI: 10.1021/acssynbio.3c00542 -
International Journal of Molecular... Jul 2023The yeast is a unique genetic object for which a wide range of relatively simple, inexpensive, and non-time-consuming methods have been developed that allow the... (Review)
Review
The yeast is a unique genetic object for which a wide range of relatively simple, inexpensive, and non-time-consuming methods have been developed that allow the performing of a wide variety of genome modifications. Among the latter, one can mention point mutations, disruptions and deletions of particular genes and regions of chromosomes, insertion of cassettes for the expression of heterologous genes, targeted chromosomal rearrangements such as translocations and inversions, directed changes in the karyotype (loss or duplication of particular chromosomes, changes in the level of ploidy), mating-type changes, etc. Classical yeast genome manipulations have been advanced with CRISPR/Cas9 technology in recent years that allow for the generation of multiple simultaneous changes in the yeast genome. In this review we discuss practical applications of both the classical yeast genome modification methods as well as CRISPR/Cas9 technology. In addition, we review methods for ploidy changes, including aneuploid generation, methods for mating type switching and directed DSB. Combined with a description of useful selective markers and transformation techniques, this work represents a nearly complete guide to yeast genome modification.
Topics: Saccharomyces cerevisiae; Gene Editing; CRISPR-Cas Systems
PubMed: 37569333
DOI: 10.3390/ijms241511960 -
Molecular Systems Biology Aug 2023The complexity of many cellular and organismal traits results from the integration of genetic and environmental factors via molecular networks. Network structure and...
The complexity of many cellular and organismal traits results from the integration of genetic and environmental factors via molecular networks. Network structure and effect propagation are best understood at the level of functional modules, but so far, no concept has been established to include the global network state. Here, we show when and how genetic perturbations lead to molecular changes that are confined to small parts of a network versus when they lead to modulation of network states. Integrating multi-omics profiling of genetically heterogeneous budding and fission yeast strains with an array of cellular traits identified a central state transition of the yeast molecular network that is related to PKA and TOR (PT) signaling. Genetic variants affecting this PT state globally shifted the molecular network along a single-dimensional axis, thereby modulating processes including energy and amino acid metabolism, transcription, translation, cell cycle control, and cellular stress response. We propose that genetic effects can propagate through large parts of molecular networks because of the functional requirement to centrally coordinate the activity of fundamental cellular processes.
Topics: Multifactorial Inheritance; Signal Transduction; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Phenotype
PubMed: 37485750
DOI: 10.15252/msb.202211493 -
The Journal of Cell Biology Aug 2023As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces...
As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis.
Topics: Cell Nucleus; Mitosis; Nuclear Envelope; Nuclear Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sumoylation; Organelle Biogenesis
PubMed: 37398994
DOI: 10.1083/jcb.202208137 -
The Biochemical Journal Oct 2023Mitophagy, the autophagic breakdown of mitochondria, is observed in eukaryotic cells under various different physiological circumstances. These can be broadly... (Review)
Review
Mitophagy, the autophagic breakdown of mitochondria, is observed in eukaryotic cells under various different physiological circumstances. These can be broadly categorized into two types: mitophagy related to quality control events and mitophagy induced during developmental transitions. Quality control mitophagy involves the lysosomal or vacuolar degradation of malfunctioning or superfluous mitochondria within lysosomes or vacuoles, and this is thought to serve as a vital maintenance function in respiring eukaryotic cells. It plays a crucial role in maintaining physiological balance, and its disruption has been associated with the progression of late-onset diseases. Developmentally induced mitophagy has been reported in the differentiation of metazoan tissues which undergo metabolic shifts upon developmental transitions, such as in the differentiation of red blood cells and muscle cells. Although the mechanistic studies of mitophagy in mammalian cells were initiated after the initial mechanistic findings in Saccharomyces cerevisiae, our current understanding of the physiological role of mitophagy in yeast remains more limited, despite the presence of better-defined assays and tools. In this review, I present my perspective on our present knowledge of mitophagy in yeast, focusing on physiological and mechanistic aspects. I aim to focus on areas where our understanding is still incomplete, such as the role of mitochondrial dynamics and the phenomenon of protein-level selectivity.
Topics: Animals; Saccharomyces cerevisiae; Mitophagy; Autophagy; Mitochondria; Saccharomyces cerevisiae Proteins; Mammals
PubMed: 37850532
DOI: 10.1042/BCJ20230279