Yeast Cell Extracts Research Articles

Using Different Types of Yeast Cell Extract, Probiotic and Abiotic to Improve Growth Performance, Carcass Characteristics and Antioxidant Activities of Broiler Chickens

Using Different Types of Yeast Cell Extract, Probiotic and Abiotic to Improve Growth Performance, Carcass Characteristics and Antioxidant Activities of Broiler Chickens

Non-Hydrolysable Analogues of Cyclic and Branched Condensed Phosphates: Chemistry and Chemical Proteomics.

Studies into the biology of condensed phosphates almost exclusively cover linear polyphosphates. However, there is evidence for the presence of cyclic polyphosphates (metaphosphates) in organisms and for enzymatic digestion of branched phosphates (ultraphosphates) with alkaline phosphatase. Further research of non-linear condensed phosphates in biology would profit from interactome data of such molecules, however, their stability in biological media is limited. Here we present syntheses of modified, non-hydrolysable analogues of cyclic and branched condensed phosphates, called meta- and ultraphosphonates, and their application in a chemical proteomics approach using yeast cell extracts. We identify putative interactors with overlapping hits for structurally related capture compounds underlining the quality of our results. The datasets serve as starting point to study the biological relevance and functions of meta- and ultraphosphates. In addition, we examine the reactivity of meta- and ultraphosphonates with implications for their "hydrolysable" analogues: Efforts to increase the ring-sizes of meta- or cyclic ultraphosphonates revealed a strong preference to form trimetaphosphate-analogue structures by cyclization and/or ring-contraction. Using carbodiimides for condensation, the so far inaccessible dianhydro product of ultraphosphonate, corresponding to P4 O11 2- , was selectively obtained and then ring-opened by different nucleophiles yielding modified cyclic ultraphosphonates.

Saccharomyces cerevisiae apurinic/apyrimidinic endonuclease 1 repairs abasic site-mediated DNA-peptide/protein cross-links

Saccharomyces cerevisiae apurinic/apyrimidinic (AP) endonuclease 1 (yApn1) is a key player of the base excision repair pathway. This multifunctional enzyme is an AP endonuclease, 3′–5′ exonuclease, 3′-phosphodiesterase, and participates in nucleotide incision repair. To the best of our knowledge, the known substrates of yApn1 are small DNA lesions such as AP sites and 3′-phospho-α,β-unsaturated aldehyde (3′-PUA). Here, we wish to report in vitro findings that yApn1 repairs bulky DNA-peptide cross-links (DpCs) and DNA-protein cross-links (DPCs) arising from AP sites and 3′-PUA. We chemically synthesized stable and linkage-defined DpCs and DPCs by oxime ligation and reductive amination, respectively. Our steady-state kinetic data showed that yApn1 repairs a 10-mer peptide-conjugated AP site and 3′-PUA with comparable efficiencies to that of processing the unconjugated lesions. We demonstrated that yApn1 is the predominant enzyme that incises AP-DpC in yeast cell extracts. We also demonstrated that yApn1 incises AP-DPCs in a DPC size-dependent manner, and prior DPC proteolysis by trypsin facilitates the repair. We further found that yApn1 removes 3′-PUA-histone DPCs with moderate efficiencies. Together, our results uncovered a novel role of yApn1 in DPC repair, and support the emerging model that proteolysis is required for efficient DPC repair.

Methods to Study Z-DNA-Induced Genetic Instability.

Alternative DNA structures that differ from the canonical B-DNA double helix, including Z-DNA, have received much attention recently due to their impact on DNA metabolic processes, including replication, transcription, and genome maintenance. Non-B-DNA-forming sequences can also stimulate genetic instability associated with disease development and evolution. Z-DNA can stimulate different types of genetic instability events in different species, and several different assays have been established to detect Z-DNA-induced DNA strand breaks and mutagenesis in prokaryotic andeukaryotic systems. In this chapter, we will introduce some of these methods including Z-DNA-induced mutation screening and detection of Z-DNA-induced strand breaks in mammalian cells, yeast, and mammalian cell extracts. Results from these assays should provide better insight into the mechanisms of Z-DNA-related genetic instability in different eukaryotic model systems.

Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics.

Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which labeling chemistry is designed. In practice, only two functional groups are present in every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative alkylation for peptide mass spectrometry and single-molecule protein sequencing that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino-acid residues for decarboxylative labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid-labeling technology in tandem to establish a new platform for fluorosequencing.

Yeast cell surface displaying VP28 antigen and its potential application for shrimp farming

VP28 is an envelope protein of White Spot Syndrome Virus (WSSV), which has been shown in previous studies to induce a high immune response in shrimp. VP28 has been produced in some host systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris as free protein. Here we showed a new strategy of anchoring VP28 on the Saccharomyces cerevisiae yeast surface and using the yeast cell extract combined with probiotic as an oral vaccine for shrimp farming. We have successfully constructed a recombinant yeast cell capable of expressing VP28 on the cell surface. The feeding diet combined with VP28 anchored yeast cell extract provided significant assurance to Litopenaeus vannamei, challenged by WSSV, resulting in a relative percent survival (RPS) of 87.10 ± 2.15%. Interestingly, the utilization of VP28 anchored yeast cell extract could enhance the efficiency of probiotic strains like Lactobacillus and Bacillus on shrimp farming. The results in both laboratory scales and field trials using extract of VP28 displaying Saccharomyces showed a growth-promoting effect in shrimp, assessed through average shrimp weight. Taken together, our results in this study demonstrated a new successful strategy of using yeast cell surface as a tool to produce VP28-based oral vaccine for shrimp aquaculture. KEY POINTS: • A new strategy of using VP28 antigen as anchored protein on S. cerevisiae yeast cell surface (S. cerevisiae::VP28) • The utilization of VP28 antigen and yeast as S. cerevisiae::VP28 extract enhanced potential protection of Litopenaeus vannamei against White Spot Syndrome Virus (RPS 87.10%) • The use of S. cerevisiae::VP28 extract increased efficiency of probiotic on shrimp growth-promoting effect either lab-scale or field trial.

Key interplay between the co-opted sorting nexin-BAR proteins and PI3P phosphoinositide in the formation of the tombusvirus replicase.

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.

The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity.

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

Harnessing PGPR inoculation through exogenous foliar application of salicylic acid and microbial extracts for improving rice growth

Plant-growth-promoting rhizobacteria (PGPR) should effectively colonize along the plant root to enhance the plantand soil health. The present investigation aims to improve the PGPR-mediated plant health benefits through above-ground foliar management. A green fluorescent protein-tagged PGPR strain, Pseudomonas chlororaphis (ZSB15-M2) was inoculated in a nonautoclaved agricultural soil before rice culturing. Salicylic acid and cell extracts of Corynebacterium glutamicum and Saccharomyces cerevisiae as a supply of hormonal and inducer compounds were applied on the foliage of the 10-days-old rice plants and subsequently observed the colonizing ability of ZSB15-M2. The cell extracts of Corynebacteria and yeast showed a 100-fold increase in the ZSB15-M2 population in the rhizosphere of rice, whereas salicylic acid had a 10-fold increase in relation to mock control. The rice root exudates collected after the spraying of salicylic acid and microbial extracts showed significantly enhanced release of total carbon, total protein, total sugar, total amino nitrogen, total nitrogen, and phenol content. In vitro assays revealed that these root exudates collected after exogenous spray of these chemicals enhanced the chemotactic motility and biofilm formation of ZSB15-M2 compared to the control plant's root exudate. Metabolomic analysis of root exudates collected from these rice plants by gas chromatography-mass spectrometry revealed that the Corynebacteria and yeast cell extracts enhanced the divergence of metabolites of rice root exudate. Further, due to these cumulative effects in the rice rhizosphere, the total chlorophyll, total protein, total nitrogen, and total phosphorus of rice were significantly improved. These observations provide insights into the rhizosphere functioning of rice plants as modulated by above-ground treatments with improved colonization of inoculant strains as well as the plant growth.

The ex planta signal activity of a Medicago ribosomal uL2 protein suggests a moonlighting role in controlling secondary rhizobial infection.

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1' whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.

Green pyomelanin-mediated synthesis of gold nanoparticles: modelling and design, physico-chemical and biological characteristics

BackgroundSynthesis of nanoparticles (NPs) and their incorporation in materials are amongst the most studied topics in chemistry, physics and material science. Gold NPs have applications in medicine due to their antibacterial and anticancer activities, in biomedical imaging and diagnostic test. Despite chemical synthesis of NPs are well characterized and controlled, they rely on the utilization of harsh chemical conditions and organic solvent and generate toxic residues. Therefore, greener and more sustainable alternative methods for NPs synthesis have been developed recently. These methods use microorganisms, mainly yeast or yeast cell extract. NPs synthesis with culture supernatants are most of the time the preferred method since it facilitates the purification scheme for the recovery of the NPs. Extraction of NPs, formed within the cells or cell-wall, is laborious, time-consuming and are not cost effective. The bioactivities of NPs, namely antimicrobial and anticancer, are known to be related to NPs shape, size and size distribution.ResultsHerein, we reported on the green synthesis of gold nanoparticles (AuNPs) mediated by pyomelanin purified from the yeast Yarrowia lipolytica. A three levels four factorial Box–Behnken Design (BBD) was used to evaluate the influence of temperature, pH, gold salt and pyomelanin concentration on the nanoparticle size distribution. Based on the BBD, a quadratic model was established and was applied to predict the experimental parameters that yield to AuNPs with specific size. The synthesized nanoparticles with median size value of 104 nm were of nanocrystalline structure, mostly polygonal or spherical. They exhibited a high colloidal stability with zeta potential of − 28.96 mV and a moderate polydispersity index of 0.267. The absence of cytotoxicity of the AuNPs was investigated on two mammalian cell lines, namely mouse fibroblasts (NIH3T3) and human osteosarcoma cells (U2OS). Cell viability was only reduced at AuNPs concentration higher than 160 µg/mL. Moreover, they did not affect on the cell morphology.ConclusionOur results indicate that different process parameters affect significantly nanoparticles size however with the mathematical model it is possible to define the size of AuNPs. Moreover, this melanin-based gold nanoparticles showed neither cytotoxicity effect nor altered cell morphology.

Influence of Glutathione on Yeast Fermentation Efficiency under Copper Stress.

Copper in grape musts can influence the fermentation efficiency of Saccharomyces cerevisiae during winemaking. The present study revealed the impact of glutathione addition on yeast strains with variable copper sensitivity. The antioxidant glutathione increased yeast vitality and fastened sugar metabolism at copper concentrations up to 0.39 mM. A significant accumulation of acetaldehyde at high copper concentrations was mitigated by the addition of 20 mg L-1 glutathione. Low recovery of glutathione added implicated a complexation of both compounds. Specific alcohol dehydrogenase (ADH) activity was inhibited or reduced in the enzyme extracts of the copper-stressed yeast cells. The activity was restored in fermentations with glutathione at a copper concentration of 0.16 mM. At low copper concentrations, glutathione decreased ADH activity presumably due to complexation of essential copper amounts. Results provide important information on the use of glutathione as an antioxidant in winemaking to counteract negative effects of copper-rich musts on copper-sensitive yeast strains.

Sensitive quantitative analysis of phosphorylated primary metabolites using selective metal oxide enrichment and GC- and IC- MS/MS

In this study, we present a novel selective cleanup/enrichment method based on metal oxide solid phase extraction combined with quantitative gas chromatography-tandem mass spectrometry and ion exchange chromatography-tandem mass spectrometry for the analysis of phosphorylated metabolites in yeast cell extracts relevant to biotechnological processes. Following screening of several commercially available metal oxide-based enrichment materials, all steps of the enrichment process (loading, washing and elution) were optimized for both the selective enrichment of 12 phosphorylated compounds from the glycolysis and pentose phosphate pathways, and the simultaneous removal of highly abundant matrix components such as organic acids and sugars. The full analytical workflow was then validated to meet the demands of accurate quantification of phosphorylated metabolites in yeast (Pichia pastoris) cell extracts using the best performing material and cleanup/enrichment method combined with quantification strategies based on internal standardization with isotopically labeled internal standards and external calibration. A good recovery (>70%) for 5 of the 12 targeted phosphorylated compounds with RSDs of less than 6.0% was obtained while many sugars, organic acids and amino acids were removed (>99% of glucose, and >95% of aspartate, succinate, glutamate, alanine, glycine, serine, threonine, proline, and valine). The use of isotopically labeled internal standards added to the samples prior to SPE, enables accurate quantification of the metabolites as it compensates for errors introduced during sample pretreatment and GC-MS or LC-MS analysis. To the best of our knowledge, this is the first time an effective and selective metal oxide-based affinity chromatography cleanup/enrichment method was designed and applied successfully for intracellular phosphorylated metabolites.

Drying Enhances Signal Intensities for Global GC⁻MS Metabolomics.

We report here that a straightforward change of the standard derivatization procedure for GC–MS metabolomics is leading to a strong increase in metabolite signal intensity. Drying samples between methoxymation and trimethylsilylation significantly increased signals by two- to tenfold in extracts of yeast cells, plant and animal tissue, and human urine. This easy step reduces the cost of sample material and the need for expensive new hardware.

Flavanols protect the yeast Saccharomyces cerevisiae against heating and freezing/thawing injury.

The aim of the study was to check whether two flavanols ((-)-epigallocatechin gallate and (+)-catechin) can ameliorate oxidative stress (OS) accompanying and contributing to the lethal effects of heating (50°C) and freezing-thawing on the yeast Saccharomyces cerevisiae. The flavanols studied increased yeast survival during heating and freezing-thawing, estimated by the colony forming assay. They improved also such indices of OS as increased production of reactive oxygen species, decrease of total antioxidant activity of yeast cell extracts and increase in the level of protein carbonyls. Amelioration of OS by flavanols increases the survival of the yeast subjected to high temperature and freezing-thawing. Flavanols may be considered as means of enhancing yeast survival under extreme temperature conditions and probably in other conditions involving OS.

P-277 - Influence of catechins on Saccharomyces cerevisiae subjected to thermal stress

Catechins (Flavan-3-ols), the main components of tea extracts, rise increasing interest due to their biological, mainly antioxidant, effects. The present study aims to investigate the capacity of selected catechins [(−)-Epigallocatechin gallate and (+)-Catechin] to protect Saccharomyces cerevisiae (two strains: wild type BY4741 and mutant BY4741Δsod1) as an eukaryotic cell model, against thermal stress promoted by a low temperature freezing at -20°C as well as by heating at a temperature of 50 °C. Oxidative stress has been demonstrated to play an important role in cell death induced by both high and low temperature. Catechins showed no toxicity to yeast cells at concentrations of up to at least 100 µM. The catechins studied, in particular (+)-Catechin and (−)-Epigallocatechin gallate, protected cells of both strains against temperature-induced lethality, as assessed by colony forming assays. The catechins (50 µM) decreased the level of reactive oxygen species in temperature-stressed yeast cells, estimated with dihydroethidine, and increased total antioxidant capacity of yeast cell extracts and elevated the level of reduced glutathione or reduced/oxidized glutathione ratio in cells exposed to -20oC, demonstrating efficient antioxidant action of catechins in the yeast model.

Mutational analysis of TAF6 revealed the essential requirement of the histone-fold domain and the HEAT repeat domain for transcriptional activation.

TAF6, bearing the histone H4-like histone-fold domain (HFD), is a subunit of the core TAF module in TFIID and SAGA transcriptional regulatory complexes. We isolated and characterized several yeast TAF6 mutants bearing amino acid substitutions in the HFD, the middle region or the HEAT repeat domain. The TAF6 mutants were highly defective for transcriptional activation by the Gcn4 and Gal4 activators. CHIP assays showed that the TAF6-HFD and the TAF6-HEAT domain mutations independently abrogated the promoter occupancy of TFIID and SAGA complex invivo. We employed genetic and biochemical assays to identify the relative contributions of the TAF6 HFD and HEAT domains. First, the temperature-sensitive phenotype of the HEAT domain mutant was suppressed by overexpression of the core TAF subunits TAF9 and TAF12, as well as TBP. The HFD mutant defect, however, was suppressed by TAF5 but not by TAF9, TAF12 or TBP. Second, the HEAT mutant but not the HFD mutant was defective for growth in the presence of transcription elongation inhibitors. Third, coimmunoprecipitation assays using yeast cell extracts indicated that the specific TAF6 HEAT domain residues are critical for the interaction of core TAF subunits with the SAGA complex but not with TFIID. The specific HFD residues in TAF6, although required for heterodimerization between TAF6 and TAF9 recombinant proteins, were dispensable for association of the core TAF subunits with TFIID and SAGA in yeast cell extracts. Taken together, the results of our studies have uncovered the non-overlapping requirement of the evolutionarily conserved HEAT domain and the HFD in TAF6 for transcriptional activation.

Soft Interactions with Model Crowders and Non-canonical Interactions with Cellular Proteins Stabilize RNA Folding

Living cells contain diverse biopolymers, creating a heterogeneous crowding environment, the impact of which on RNA folding is poorly understood. Here, we have used single-molecule fluorescence resonance energy transfer to monitor tertiary structure formation of the hairpin ribozyme as a model to probe the effects of polyethylene glycol and yeast cell extract as crowding agents. As expected, polyethylene glycol stabilizes the docked, catalytically active state of the ribozyme, in part through excluded volume effects; unexpectedly, we found evidence that it additionally displays soft, non-specific interactions with the ribozyme. Yeast extract has a profound effect on folding at protein concentrations 1000-fold lower than found intracellularly, suggesting the dominance of specific interactions over volume exclusion. Gel shift assays and affinity pull-down followed by mass spectrometry identified numerous non-canonical RNA-binding proteins that stabilize ribozyme folding; the apparent chaperoning activity of these ubiquitous proteins significantly compensates for the low-counterion environment of the cell.

Comparison of fully wettable RPLC stationary phases for LC-MS-based cellular metabolomics.

Reversed-phase LC combined with high-resolution mass spectrometry (HRMS) is one of the most popular methods for cellular metabolomics studies. Due to the difficulties in analyzing a wide range of polarities encountered in the metabolome, 100%-wettable reversed-phase materials are frequently used to maximize metabolome coverage within a single analysis. Packed with silica-based sub-3μm diameter particles, these columns allow high separation efficiency and offer a reasonable compromise for metabolome coverage within a single analysis. While direct performance comparison can be made using classical chromatographic characterization approaches, a comprehensive assessment of the column's performance for cellular metabolomics requires use of a full LC-HRMS workflow in order to reflect realistic study conditions used for cellular metabolomics. In this study, a comparison of several reversed-phase LC columns for metabolome analysis using such a dedicated workflow is presented. All columns were tested under the same analytical conditions on an LC-TOF-MS platform using a variety of authentic metabolite standards and biotechnologically relevant yeast cell extracts. Data on total workflow performance including retention behavior, peak capacity, coverage, and molecular feature extraction repeatability from these columns are presented with consideration for both nontargeted screening and differential metabolomics workflows using authentic standards and Pichia pastoris cell extract samples.

Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

SummaryDisassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.