Bait Protein Research Articles

The promiscuous biotin ligase TurboID reveals the proxisome of the T3SS chaperone IpgC in Shigella flexneri.

Promiscuous biotin ligases derived from the bacterial enzyme BirA are used to identify proteins vicinal to a bait protein, thereby defining its proxisome. Despite the popularity of this approach, surprisingly little is known about its use in prokaryotes. Here, we compared the activity of four widely used promiscuous biotin ligases in the cytoplasm of Shigella flexneri, a pathogenic subgroup of Escherichia coli. Our data indicate that the kinetics of TurboID's biotinylating activity is the highest of those tested. In addition, TurboID showed reduced interaction with the natural BirA binding partners, BccP and the biotin operator, when compared to its ancestor BioID. We therefore evaluated the ability of TurboID to probe the proxisome of the type III secretion system (T3SS) chaperone IpgC and the transcriptional activator MxiE. When the T3SS is inactive (off-state), these proteins are inhibited by forming complexes with the T3SS substrates OspD1 and IpaBC, respectively. In contrast, when the T3SS is active (on-state), OspD1 and IpaBC are secreted allowing MxiE and IpgC to interact together and activate their target genes. The results obtained with the IpgC and TurboID fusions capture a good fraction of these known interactions. It also suggests that the availability of IpgC increases in the on-state, resulting in a greater number of proteins detected in its vicinity. Among these is the T3SS ATPase SpaL (also known as Spa47 or SctN), further supporting the notion that chaperones escort their substrate to the T3SS. Interestingly, a specific subset of proteins conserved in E. coli completes the IpgC proxisome in the on-state.IMPORTANCEPromiscuous biotin ligases are widely used to study protein function in eukaryotes. Strikingly, their use in prokaryotes has been rare. Indeed, the small volume and the cytoplasmic location of the biotin ligase's natural binding partners in these organisms pose unique challenges that can interfere with the study of the proxisome of proteins of interest. Here, we evaluated four of the most common promiscuous biotin ligases and found TurboID was best suited for use in the cytoplasm of Shigella flexneri. Using this method, we extended the proxisome of IpgC beyond its known direct binding partners involved in the regulation of the type III secretion system (T3SS) signaling cascade. Of particular interest for further study are transcription factors and housekeeping proteins that are enriched around IpgC when the T3SS is active. We propose a model in which the increased availability of IpgC in the on-state may allow cross-talk of the T3SS with other cellular processes.

Imaging the Raf-MEK-ERK Signaling Cascade in Living Cells.

Conventional biochemical methods for studying cellular signaling cascades have relied on destructive cell disruption. In contrast, the live cell imaging of fluorescent-tagged transfected proteins offers a non-invasive approach to understanding signal transduction events. One strategy involves monitoring the phosphorylation-dependent shuttling of a fluorescent-labeled kinase between the nucleus and cytoplasm using nuclear localization, export signals, or both. In this paper, we introduce a simple method to visualize intracellular signal transduction in live cells by exploring the translocation properties of PKC from the cytoplasm to the membrane. We fused bait protein to PKC, allowing the bait (RFP-labeled) and target (GFP-labeled) proteins to co-translocate from the cytoplasm to the membrane. However, in non-interacting protein pairs, only the bait protein was translocated to the plasma membrane. To verify our approach, we examined the Raf-MEK-ERK signaling cascade (ERK pathway). We successfully visualized direct Raf1/MEK2 interaction and the KSR1-containing ternary complex (Raf1/MEK2/KSR1). However, the interaction between MEK and ERK was dependent on the presence of the KSR1 scaffold protein under our experimental conditions.

DEAD-box RNA helicase RH20 positively regulates RNAi-based antiviral immunity in plants by associating with SGS3/RDR6 bodies.

RNA silencing plays a crucial role in defending against viral infections in diverse eukaryotic hosts. Despite extensive studies on core components of the antiviral RNAi pathway such as DCLs, AGOs and RDRs proteins, host factors involved in antiviral RNAi remain incompletely understood. In this study, we employed the proximity labelling approach to identify the host factors required for antiviral RNAi in Nicotiana benthamiana. Using the barley stripe mosaic virus (BSMV)-encoded γb, a viral suppressor of RNA silencing (VSR), as the bait protein, we identified the DEAD-box RNA helicase RH20, a broadly conserved protein in plants and animals with a homologous human protein known as DDX5. We demonstrated the interaction between RH20 and BSMV γb. Knockdown or knockout of RH20 attenuates the accumulation of viral small interfering RNAs, leading to increased susceptibility to BSMV, while overexpression of RH20 enhances resistance to BSMV, a process requiring the cytoplasmic localization and RNA-binding activity of RH20. In addition to BSMV, RH20 also negatively regulates the infection of several other positive-sense RNA viruses, suggesting the broad-spectrum antiviral activity of RH20. Mechanistic analysis revealed the colocalization and interaction of RH20 with SGS3/RDR6, and disruption of either SGS3 or RDR6 undermines the antiviral function of RH20, suggesting RH20 as a new component of the SGS3/RDR6 bodies. As a counter-defence, BSMV γb VSR subverts the RH20-mediated antiviral defence by interfering with the RH20-SGS3 interaction. Our results uncover RH20 as a new positive regulator of antiviral RNAi and provide new potential targets for controlling plant viral diseases.

Detection of differential bait proteoforms through immunoprecipitation-mass spectrometry data analysis

Proteins are often referred to as the workhorses of cells, and their interactions are necessary to facilitate specific cellular functions. Despite the recognition that protein-protein interactions, and thus protein functions, are determined by proteoform states, such as mutations and post-translational modifications (PTMs), methods for determining the differential abundance of proteoforms across conditions are very limited. Classically, immunoprecipitation coupled with mass spectrometry (IP-MS) has been used to understand how the interactome (preys) of a given protein (bait) changes between conditions to elicit specific cellular functions. Reversing this concept, we present here a new workflow for IP-MS data analysis that focuses on identifying the differential peptidoforms of the bait protein between conditions. This method can provide detailed information about specific bait proteoforms, potentially revealing pathogenic protein states that can be exploited for the development of targeted therapies.

A comparative analysis of paxillin and Hic-5 proximity interactomes.

Focal adhesions serve as structural and signaling hubs, facilitating bidirectional communication at the cell-extracellular matrix interface. Paxillin and the related Hic-5 (TGFβ1i1) are adaptor/scaffold proteins that recruit numerous structural and regulatory proteins to focal adhesions, where they perform both overlapping and discrete functions. In this study, paxillin and Hic-5 were expressed in U2OS osteosarcoma cells as biotin ligase (BioID2) fusion proteins and used as bait proteins for proximity-dependent biotinylation in order to directly compare their respective interactomes. The fusion proteins localized to both focal adhesions and the centrosome, resulting in biotinylation of components of each of these structures. Biotinylated proteins were purified and analyzed by mass spectrometry. The list of proximity interactors for paxillin and Hic-5 comprised numerous shared core focal adhesion proteins that likely contribute to their similar functions in cell adhesion and migration, as well as proteins unique to paxillin and Hic-5 that have been previously localized to focal adhesions, the centrosome, or the nucleus. Western blotting confirmed biotinylation and enrichment of FAK and vinculin, known interactors of Hic-5 and paxillin, as well as several potentially unique proximity interactors of Hic-5 and paxillin, including septin 7 and ponsin, respectively. Further investigation into the functional relationship between the unique interactors and Hic-5 or paxillin may yield novel insights into their distinct roles in cell migration.

The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein–protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

DIP-MS: ultra-deep interaction proteomics for the deconvolution of protein complexes

Most proteins are organized in macromolecular assemblies, which represent key functional units regulating and catalyzing most cellular processes. Affinity purification of the protein of interest combined with liquid chromatography coupled to tandem mass spectrometry (AP–MS) represents the method of choice to identify interacting proteins. The composition of complex isoforms concurrently present in the AP sample can, however, not be resolved from a single AP–MS experiment but requires computational inference from multiple time- and resource-intensive reciprocal AP–MS experiments. Here we introduce deep interactome profiling by mass spectrometry (DIP-MS), which combines AP with blue-native-PAGE separation, data-independent acquisition with mass spectrometry and deep-learning-based signal processing to resolve complex isoforms sharing the same bait protein in a single experiment. We applied DIP-MS to probe the organization of the human prefoldin family of complexes, resolving distinct prefoldin holo- and subcomplex variants, complex–complex interactions and complex isoforms with new subunits that were experimentally validated. Our results demonstrate that DIP-MS can reveal proteome modularity at unprecedented depth and resolution.

Tip Growth Defective1 interacts with the cellulose synthase complex to regulate cellulose synthesis in Arabidopsis thaliana.

Plant cells possess robust and flexible cell walls composed primarily of cellulose, a polysaccharide that provides structural support and enables cell expansion. Cellulose is synthesised by the Cellulose Synthase A (CESA) catalytic subunits, which form cellulose synthase complexes (CSCs). While significant progress has been made in unravelling CSC function, the trafficking of CSCs and the involvement of post-translational modifications in cellulose synthesis remain poorly understood. In order to deepen our understanding of cellulose biosynthesis, this study utilised immunoprecipitation techniques with CESA6 as the bait protein to explore the CSC and its interactors. We have successfully identified the essential components of the CSC complex and, notably, uncovered novel interactors associated with CSC trafficking, post-translational modifications, and the coordination of cell wall synthesis. Moreover, we identified TIP GROWTH DEFECTIVE 1 (TIP1) protein S-acyl transferases (PATs) as an interactor of the CSC complex. We confirmed the interaction between TIP1 and the CSC complex through multiple independent approaches. Further analysis revealed that tip1 mutants exhibited stunted growth and reduced levels of crystalline cellulose in leaves. These findings suggest that TIP1 positively influences cellulose biosynthesis, potentially mediated by its role in the S-acylation of the CSC complex.

Interaction network of human early embryonic transcription factors

Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.

The RNA helicase Dbp10 coordinates assembly factor association with PTC maturation during ribosome biogenesis.

During ribosome biogenesis a plethora of assembly factors and essential enzymes drive the unidirectional maturation of nascent pre-ribosomal subunits. The DEAD-box RNA helicase Dbp10 is suggested to restructure pre-ribosomal rRNA of the evolving peptidyl-transferase center (PTC) on nucleolar ribosomal 60S assembly intermediates. Here, we show that point mutations within conserved catalytic helicase-core motifs of Dbp10 yield a dominant-lethal growth phenotype. Such dbp10 mutants, which stably associate with pre-60S intermediates, impair pre-60S biogenesis at a nucleolar stage prior to the release of assembly factor Rrp14 and stable integration of late nucleolar factors such as Noc3. Furthermore, the binding of the GTPase Nug1 to particles isolated directly via mutant Dbp10 bait proteins is specifically inhibited. The N-terminal domain of Nug1 interacts with Dbp10 and the methyltransferase Spb1, whose pre-60S incorporation is also reduced in absence of functional Dbp10 resulting in decreased methylation of 25S rRNA nucleotide G2922. Our data suggest that Dbp10's helicase activity generates the necessary framework for assembly factor docking thereby permitting PTC rRNA methylation and the progression of pre-60S maturation.

TurboID-mediated proximity labeling for screening interacting proteins of FIP37 in Arabidopsis.

Proximity labeling was recently developed to detect protein-protein interactions and members of subcellular multiprotein structures in living cells. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity, such as biotin ligase, to a protein of interest (bait protein) to biotinylate adjacent proteins. The biotinylated protein can be purified by streptavidin beads, and identified by mass spectrometry (MS). TurboID is an engineered biotin ligase with high catalytic efficiency, which is used for proximity labeling. Although TurboID-based proximity labeling technology has been successfully established in mammals, its application in plant systems is limited. Here, we report the usage of TurboID for proximity labeling of FIP37, a core member of m6A methyltransferase complex, to identify FIP37 interacting proteins in Arabidopsis thaliana. By analyzing the MS data, we found 214 proteins biotinylated by GFP-TurboID-FIP37 fusion, including five components of m6A methyltransferase complex that have been previously confirmed. Therefore, the identified proteins may include potential proteins directly involved in the m6A pathway or functionally related to m6A-coupled mRNA processing due to spatial proximity. Moreover, we demonstrated the feasibility of proximity labeling technology in plant epitranscriptomics study, thereby expanding the application of this technology to more subjects of plant research.

Screening of Tnfaip1-Interacting Proteins in Zebrafish Embryonic cDNA Libraries Using a Yeast Two-Hybrid System.

TNFAIP1 regulates cellular biological functions, including DNA replication, DNA repair, and cell cycle, by binding to target proteins. Identification of Tnfaip1-interacting proteins contributes to the understanding of the molecular regulatory mechanisms of their biological functions. In this study, 48 hpf, 72 hpf, and 96 hpf wild-type zebrafish embryo mRNAs were used to construct yeast cDNA library. The library titer was 1.12 × 107 CFU/mL, the recombination rate was 100%, and the average length of the inserted fragments was greater than 1000 bp. A total of 43 potential interacting proteins of Tnfaip1 were identified using zebrafish Tnfaip1 as a bait protein. Utilizing GO functional annotation and KEGG signaling pathway analysis, we found that these interacting proteins are mainly involved in translation, protein catabolic process, ribosome assembly, cytoskeleton formation, amino acid metabolism, and PPAR signaling pathway. Further yeast spotting analyses identified four interacting proteins of Tnfaip1, namely, Ubxn7, Tubb4b, Rpl10, and Ybx1. The Tnfaip1-interacting proteins, screened from zebrafish embryo cDNA in this study, increased our understanding of the network of Tnfaip1-interacting proteins during the earliest embryo development and provided a molecular foundation for the future exploration of tnfaip1's biological functions.

Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification-Mass Spectrometry.

We present an effective, fast, and user-friendly method to reduce codigestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short preincubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using nontoxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC-MS analysis, improving sensitivity and quantitative accuracy.

Proximity labeling and identification of endogenous client proteins recruited to Y15-based artificial granules tethering a bait protein.

Protein clustering is a ubiquitous event in diverse cellular processes. Self-association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15-based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1-tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC-MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.

A conserved STRIPAK complex is required for autophagy in muscle tissue.

Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in-silico approach, we identified Striatin interacting protein (Strip), MOB kinase activator 4, and fibroblast growth factor receptor 1 oncogene partner 2 as conserved mediators of muscle tissue maintenance. We performed affinity purification-mass spectrometry (AP-MS) experiments with Drosophila melanogaster Strip as a bait protein and copurified additional Striatin-interacting phosphatase and kinase (STRIPAK) complex members from larval muscle tissue. NUAK family kinase 1 (NUAK) and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using proximity ligation assays. To understand the functional significance of the STRIPAK-NUAK-Stv complex, we employed a sensitized genetic assay combined with RNA interference (RNAi) to demonstrate that both NUAK and stv function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of Strip in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Autophagy-related 8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in Strip RNAi muscles, while lysosome biogenesis and activity were unaffected. Our results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue.

ELISA-Based Method to Analyze Interactions between Bait and Prey Proteins

ELISA-Based Method to Analyze Interactions between Bait and Prey Proteins

Control of TurboID-dependent biotinylation intensity in proximity ligation screens

Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system's inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilized avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions. SignificanceProximity biotinylation screens using the last generation biotin ligase TurboID represent a powerful approach for the characterisation of transient protein-protein interaction and signaling networks. However, a constant and high basal biotinylation rate and the associated cytotoxicity often preclude the use of this method in proteomic studies. We report a protocol based on modulation of free biotin levels that prevents the deleterious effects of TurboID while allowing inducible biotinylation, even in subcellular compartments such as the endoplasmic reticulum. This optimised protocol greatly expands the applications of TurboID in proteomic screens.

Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.

Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and regulation. Proximity biotinylation has emerged as a highly specific approach to capture transient and stable interactions in living cells or organisms. Proximity biotinylation exploits promiscuous biotinylating enzymes fused to a bait protein, resulting in the biotinylation of adjacent endogenous proteins. Biotinylated interactors are purified under very strict conditions and identified by mass spectrometry to obtain a high-confidence list of candidate binding partners. AirID is a recently described biotin ligase specifically engineered for proximity labeling. This protocol details proximity biotinylation by AirID, using protein complexes that form during a type I interferon response as an example. It covers the construction and validation of AirID fusion proteins and the enrichment and identification of biotinylated interactors. We describe a variation on the protocol using splitAirID. In this case, AirID is split into two inactive fragments and ligase activity is only restored upon dimerization of the bait proteins. This permits selective detection of proteins that interact with homo- or heterodimeric forms of the bait. The protocol considers design strategies, optimization, and the properties of different biotin ligases to identify optimal conditions for each experimental question. We also discuss common pitfalls and how to troubleshoot them. These approaches allow proximity biotinylation to be a powerful tool for defining protein interactomes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction and functional validation of AirID fusion proteins Alternate Protocol: Construction and functional validation of splitAirID fusion proteins Support Protocol: Western blot for biotinylated proteins Basic Protocol 2: Biotinylation, enrichment, and identification of protein interactors.

TEAD Proteins Associate With DNA Repair Proteins to Facilitate Cellular Recovery From DNA Damage

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage–induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases)

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (∼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.