Structural basis of mitochondrial protein import by the TIM23 complex.

Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope1-5. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel6-11. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.

Massive expression of cysteine-containing proteins causes abnormal elongation of yeast cells by perturbing the proteasome.

The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins.

Engineered protein-small molecule conjugates empower selective enzyme inhibition.

Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

Phosphate Restriction Promotes Longevity via Activation of Autophagy and the Multivesicular Body Pathway.

Nutrient limitation results in an activation of autophagy in organisms ranging from yeast, nematodes and flies to mammals. Several evolutionary conserved nutrient-sensing kinases are critical for efficient adaptation of yeast cells to glucose, nitrogen or phosphate depletion, subsequent cell-cycle exit and the regulation of autophagy. Here, we demonstrate that phosphate restriction results in a prominent extension of yeast lifespan that requires the coordinated activity of autophagy and the multivesicular body pathway, enabling efficient turnover of cytoplasmic and plasma membrane cargo. While the multivesicular body pathway was essential during the early days of aging, autophagy contributed to long-term survival at later days. The cyclin-dependent kinase Pho85 was critical for phosphate restriction-induced autophagy and full lifespan extension. In contrast, when cell-cycle exit was triggered by exhaustion of glucose instead of phosphate, Pho85 and its cyclin, Pho80, functioned as negative regulators of autophagy and lifespan. The storage of phosphate in form of polyphosphate was completely dispensable to in sustaining viability under phosphate restriction. Collectively, our results identify the multifunctional, nutrient-sensing kinase Pho85 as critical modulator of longevity that differentially coordinates the autophagic response to distinct kinds of starvation.

Tubulin isotypes optimize distinct spindle positioning mechanisms during yeast mitosis.

Microtubules are dynamic cytoskeleton filaments that are essential for a wide range of cellular processes. They are polymerized from tubulin, a heterodimer of α- and ß-subunits. Most eukaryotic organisms express multiple isotypes of α- and ß-tubulin, yet their functional relevance in any organism remains largely obscure. The two α-tubulin isotypes in budding yeast, Tub1 and Tub3, are proposed to be functionally interchangeable, yet their individual functions have not been rigorously interrogated. Here, we develop otherwise isogenic yeast strains expressing single tubulin isotypes at levels comparable to total tubulin in WT cells. Using genome-wide screening, we uncover unique interactions between the isotypes and the two major mitotic spindle positioning mechanisms. We further exploit these cells to demonstrate that Tub1 and Tub3 optimize spindle positioning by differentially recruiting key components of the Dyn1- and Kar9-dependent mechanisms, respectively. Our results provide novel mechanistic insights into how tubulin isotypes allow highly conserved microtubules to function in diverse cellular processes.

Meiosis initiation: a story of two sexes in all creatures great and small.

Meiosis facilitates diversity across individuals and serves as a major driver of evolution. However, understanding how meiosis begins is complicated by fundamental differences that exist between sexes and species. Fundamental meiotic research is further hampered by a current lack of human meiotic cells lines. Consequently, much of what we know relies on data from model organisms. However, contextualising findings from yeast, worms, flies and mice can be challenging, due to marked differences in both nomenclature and the relative timing of meiosis. In this review, we set out to combine current knowledge of signalling and transcriptional pathways that control meiosis initiation across the sexes in a variety of organisms. Furthermore, we highlight the emerging links between meiosis initiation and oncogenesis, which might explain the frequent re-expression of normally silent meiotic genes in a variety of human cancers.

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Selective increment of phosphatidylserine on the autophagic body membrane in the yeast vacuole.

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.

Extracts of Digested Berries Increase the Survival of Saccharomyces cerevisiae during H2O2 Induced Oxidative Stress.

Many studies suggest anthocyanins may prevent the development of several diseases. However, anthocyanin bioactivity against cellular stress is not fully understood. This study aimed to evaluate the protective effect of berry anthocyanins on stressed cells using Saccharomyces cerevisiae. The impact of in vitro gastrointestinal digestion on anthocyanin profiles was also assessed. Bilberry and blackcurrant had higher anthocyanin levels than raspberry and strawberry, but digestion reduced the detected anthocyanins by approximately 90%. Yeast cells with and without digested or nondigested anthocyanin extracts were exposed to H2O2 and examined for survival. In the presence of anthocyanins, particularly from digested strawberry, a significant increase in cell survival was observed, suggesting that the type and levels of anthocyanins are important factors, but they also need to undergo gastrointestinal (GI) structural modifications to induce cell defence. Results also showed that cells need to be exposed to anthocyanins before the stress was applied, suggesting induction of a cellular defence system by anthocyanins or their derivatives rather than by a direct antioxidative effect on H2O2. Overall, data showed that exposure of severely stressed yeast cells to digested berry extracts improved cell survival. The findings also showed the importance of considering gastrointestinal digestion when evaluating anthocyanins' biological activity.

Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex.

Previous work has suggested that highly positively charged protein segments coded by rare codons or poly (A) stretches induce ribosome stalling and translational arrest through electrostatic interactions with the negatively charged ribosome exit tunnel, leading to inefficient elongation. This arrest leads to the activation of the Ribosome Quality Control (RQC) pathway and results in low expression of these reporter proteins. However, the only endogenous yeast proteins known to activate the RQC are Rqc1, a protein essential for RQC function, and Sdd1, a protein with unknown function, both of which contain polybasic sequences. To explore the generality of this phenomenon, we investigated whether the RQC complex controls the expression of other proteins with polybasic sequences. We showed by ribosome profiling data analysis and western blot that proteins containing polybasic sequences similar to, or even more positively charged than those of Rqc1 and Sdd1, were not targeted by the RQC complex. We also observed that the previously reported Ltn1-dependent regulation of Rqc1 is posttranslational, independent of the RQC activity. Taken together, our results suggest that RQC should not be regarded as a general regulatory pathway for the expression of highly positively charged proteins in yeast.

Loop extrusion as a mechanism for formation of DNA damage repair foci.

The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (γH2AX), which act as seeds for the formation of DNA-damage response foci1. It is unclear how these foci are rapidly assembled to establish a 'repair-prone' environment within the nucleus. Topologically associating domains are a key feature of 3D genome organization that compartmentalize transcription and replication, but little is known about their contribution to DNA repair processes2,3. Here we show that topologically associating domains are functional units of the DNA damage response, and are instrumental for the correct establishment of γH2AX-53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model in which H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the importance of chromosome conformation in the maintenance of genome integrity and demonstrates the establishment of a chromatin modification by loop extrusion.

Biological autoluminescence as a noninvasive monitoring tool for chemical and physical modulation of oxidation in yeast cell culture.

Normal or excessive oxidative metabolism in organisms is essential in physiological and pathophysiological processes, respectively. Therefore, monitoring of biological oxidative processes induced by the chemical or physical stimuli is nowadays of extreme importance due to the environment overloaded with various physicochemical factors. Current techniques typically require the addition of chemical labels or light illumination, which perturb the samples to be analyzed. Moreover, the current techniques are very demanding in terms of sample preparation and equipment. To alleviate these limitations, we propose a label-free monitoring tool of oxidation based on biological autoluminescence (BAL). We demonstrate this tool on Saccharomyces cerevisiae cell culture. We showed that BAL can be used to monitor chemical perturbation of yeast due to Fenton reagents initiated oxidation-the BAL intensity changes with hydrogen peroxide concentration in a dose-dependent manner. Furthermore, we also showed that BAL reflects the effects of low-frequency magnetic field on the yeast cell culture, where we observed a disturbance of the BAL kinetics in the exposed vs. control case. Our results contribute to the development of novel techniques for label-free, real-time, noninvasive monitoring of oxidative processes and approaches for their modulation.

Autophagy sustains glutamate and aspartate synthesis in Saccharomyces cerevisiae during nitrogen starvation.

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

Bulk autophagy induction and life extension is achieved when iron is the only limited nutrient in Saccharomyces cerevisiae.

We have investigated the effects that iron limitation provokes in Saccharomyces cerevisiae exponential cultures. We have demonstrated that one primary response is the induction of bulk autophagy mediated by TORC1. Coherently, Atg13 became dephosphorylated whereas Atg1 appeared phosphorylated. The signal of iron deprivation requires Tor2/Ypk1 activity and the inactivation of Tor1 leading to Atg13 dephosphorylation, thus triggering the autophagy process. Iron replenishment in its turn, reduces autophagy flux through the AMPK Snf1 and the subsequent activity of the iron-responsive transcription factor, Aft1. This signalling converges in Atg13 phosphorylation mediated by Tor1. Iron limitation promotes accumulation of trehalose and the increase in stress resistance leading to a quiescent state in cells. All these effects contribute to the extension of the chronological life, in a manner totally dependent on autophagy activation.

Tracking break-induced replication shows that it stalls at roadblocks.

Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases1,2. Previous studies have defined the enzymes that are required for BIR1-5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR progression, and BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and how BIR contributes to genome instability.

Selection for rapid uptake of scarce or fluctuating resource explains vulnerability of glycolysis to imbalance.

Glycolysis is a conserved central pathway in energy metabolism that converts glucose to pyruvate with net production of two ATP molecules. Because ATP is produced only in the lower part of glycolysis (LG), preceded by an initial investment of ATP in the upper glycolysis (UG), achieving robust start-up of the pathway upon activation presents a challenge: a sudden increase in glucose concentration can throw a cell into a self-sustaining imbalanced state in which UG outpaces LG, glycolytic intermediates accumulate and the cell is unable to maintain high ATP concentration needed to support cellular functions. Such metabolic imbalance can result in "substrate-accelerated death", a phenomenon observed in prokaryotes and eukaryotes when cells are exposed to an excess of substrate that previously limited growth. Here, we address why evolution has apparently not eliminated such a costly vulnerability and propose that it is a manifestation of an evolutionary trade-off, whereby the glycolysis pathway is adapted to quickly secure scarce or fluctuating resource at the expense of vulnerability in an environment with ample resource. To corroborate this idea, we perform individual-based eco-evolutionary simulations of a simplified yeast glycolysis pathway consisting of UG, LG, phosphate transport between a vacuole and a cytosol, and a general ATP demand reaction. The pathway is evolved in constant or fluctuating resource environments by allowing mutations that affect the (maximum) reaction rate constants, reflecting changing expression levels of different glycolytic enzymes. We demonstrate that under limited constant resource, populations evolve to a genotype that exhibits balanced dynamics in the environment it evolved in, but strongly imbalanced dynamics under ample resource conditions. Furthermore, when resource availability is fluctuating, imbalanced dynamics confers a fitness advantage over balanced dynamics: when glucose is abundant, imbalanced pathways can quickly accumulate the glycolytic intermediate FBP as intracellular storage that is used during periods of starvation to maintain high ATP concentration needed for growth. Our model further predicts that in fluctuating environments, competition for glucose can result in stable coexistence of balanced and imbalanced cells, as well as repeated cycles of population crashes and recoveries that depend on such polymorphism. Overall, we demonstrate the importance of ecological and evolutionary arguments for understanding seemingly maladaptive aspects of cellular metabolism.

The Effect of Dia2 Protein Deficiency on the Cell Cycle, Cell Size, and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae.

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCFDia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4-an essential DNA replication protein and substrate of Dia2-prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.

Fifty years of cycling.

Fifty years ago, the first isolation of conditional budding yeast mutants that were defective in cell division was reported. Looking back, we now know that the analysis of these mutants revealed the molecular mechanisms and logic of the cell cycle, identified key regulatory enzymes that drive the cell cycle, elucidated structural components that underly essential cell cycle processes, and influenced our thinking about cancer and other diseases. Here, we briefly summarize what was concluded about the coordination of the cell cycle 50 years ago and how that relates to our current understanding of the molecular events that have since been elucidated.

Interaction between Polyphenolic Antioxidants and Saccharomyces cerevisiae Cells Defective in Heavy Metal Transport across the Plasma Membrane.

Natural polyphenols are compounds with important biological implications which include antioxidant and metal-chelating characteristics relevant for their antimicrobial, antitumor, or antiaging potential. The mechanisms linking polyphenols and heavy metals in their concerted actions on cells are not completely elucidated. In this study, we used the model eukaryotic microorganism Saccharomyces cerevisiae to detect the action of widely prevalent natural polyphenols on yeast cells defective in the main components involved in essential heavy metal transport across the plasma membrane. We found that caffeic and gallic acids interfered with Zn accumulation, causing delays in cell growth that were alleviated by Zn supplementation. The flavones morin and quercetin interfered with both Mn and Zn accumulation, which resulted in growth improvement, but supplemental Mn and especially Zn turned the initially benefic action of morin and quercetin into potential toxicity. Our results imply that caution is needed when administering food supplements or nutraceuticals which contain both natural polyphenols and essential elements, especially zinc.

Cellular Control of Viscosity Counters Changes in Temperature and Energy Availability.

Cellular functioning requires the orchestration of thousands of molecular interactions in time and space. Yet most molecules in a cell move by diffusion, which is sensitive to external factors like temperature. How cells sustain complex, diffusion-based systems across wide temperature ranges is unknown. Here, we uncover a mechanism by which budding yeast modulate viscosity in response to temperature and energy availability. This "viscoadaptation" uses regulated synthesis of glycogen and trehalose to vary the viscosity of the cytosol. Viscoadaptation functions as a stress response and a homeostatic mechanism, allowing cells to maintain invariant diffusion across a 20°C temperature range. Perturbations to viscoadaptation affect solubility and phase separation, suggesting that viscoadaptation may have implications for multiple biophysical processes in the cell. Conditions that lower ATP trigger viscoadaptation, linking energy availability to rate regulation of diffusion-controlled processes. Viscoadaptation reveals viscosity to be a tunable property for regulating diffusion-controlled processes in a changing environment.