The Proteomic Composition and Organization of Constitutive Heterochromatin in Mouse Tissues.

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo.

Microtubule association of TRIM3 revealed by differential extraction proteomics.

The microtubule network is formed from polymerised tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule-binding proteins, we have developed an unbiased biochemical assay, which relies on the selective extraction of cytosolic proteins from U2OS cells, while leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug nocodazole or the microtubule-stabilising drug taxol, which is quantitated by mass spectrometry. Our approach is benchmarked by co-segregation of tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule-binding proteins, from which we have selected the ubiquitin E3 ligase tripartite motif-containing protein 3 (TRIM3) for further characterisation. We map TRIM3 microtubule binding to its C-terminal NHL-repeat region. We show that TRIM3 is required for the accumulation of acetylated tubulin, following treatment with taxol. Furthermore, loss of TRIM3 partially recapitulates the reduction in nocodazole-resistant microtubules characteristic of α-tubulin acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 following depletion of TRIM3 that is independent of transcription.

GIPC1 regulates MACC1-driven metastasis.

Background: Identification of cancer metastasis-relevant molecular networks is desired to provide the basis for understanding and developing intervention strategies. Here we address the role of GIPC1 in the process of MACC1-driven metastasis. MACC1 is a prognostic indicator for patient metastasis formation and metastasis-free survival. MACC1 controls gene transcription, promotes motility, invasion and proliferation of colon cancer cells in vitro, and causes tumor growth and metastasis in mice. Methods: By using yeast-two-hybrid assay, mass spectrometry, co-immunoprecipitation and peptide array we analyzed GIPC1 protein binding partners, by using the MACC1 gene promoter and chromatin immunoprecipitation and electrophoretic mobility shift assay we probed for GIPC1 as transcription factor. We employed GIPC1/MACC1-manipulated cell lines for in vitro and in vivo analyses, and we probed the GIPC1/MACC1 impact using human primary colorectal cancer (CRC) tissue. Results: We identified MACC1 and its paralogue SH3BP4 as protein binding partners of the protein GIPC1, and we also demonstrated the binding of GIPC1 as transcription factor to the MACC1 promoter (TSS to -60 bp). GIPC1 knockdown reduced endogenous, but not CMV promoter-driven MACC1 expression, and diminished MACC1-induced cell migration and invasion. GIPC1 suppression reduced tumor growth and metastasis in mice intrasplenically transplanted with MACC1-overexpressing CRC cells. In human primary CRC specimens, GIPC1 correlates with MACC1 expression and is of prognostic value for metastasis formation and metastasis-free survival. Combination of MACC1 and GIPC1 expression improved patient survival prognosis, whereas SH3BP4 expression did not show any prognostic value. Conclusions: We identified an important, dual function of GIPC1 - as protein interaction partner and as transcription factor of MACC1 - for tumor progression and cancer metastasis.

Stable Isotope-Assisted Untargeted Metabolomics Identifies ALDH1A1-Driven Erythronate Accumulation in Lung Cancer Cells.

Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer.

Using PrISMa to reveal the interactome of the human claudins family.

Here, we provide a protocol for the systematic screening of protein-protein interactions mediated by short linear motifs using the Protein Interaction Screen on a peptide Matrix (PrISMa) technique. We describe how to pull down interacting proteins in a parallelized manner and identify them by mass spectrometry. Finally, we describe a bioinformatic workflow necessary to identify highly probable interaction partners in the large-scale dataset. We describe the application of this method for the transient interactome of the claudin protein family. For complete details on the use and execution of this protocol, please refer to Suarez-Artiles et al.1.

Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed.

Queuosine (Q) is a modified nucleoside at the wobble position of specific tRNAs. In mammals, queuosinylation is facilitated by queuine uptake from the gut microbiota and is introduced into tRNA by the QTRT1-QTRT2 enzyme complex. By establishing a Qtrt1 knockout mouse model, we discovered that the loss of Q-tRNA leads to learning and memory deficits. Ribo-Seq analysis in the hippocampus of Qtrt1-deficient mice revealed not only stalling of ribosomes on Q-decoded codons, but also a global imbalance in translation elongation speed between codons that engage in weak and strong interactions with their cognate anticodons. While Q-dependent molecular and behavioral phenotypes were identified in both sexes, female mice were affected more severely than males. Proteomics analysis confirmed deregulation of synaptogenesis and neuronal morphology. Together, our findings provide a link between tRNA modification and brain functions and reveal an unexpected role of protein synthesis in sex-dependent cognitive performance.

Docking and stability defects in mitofusin highlight the proteasome as a potential therapeutic target.

Defects in mitochondrial fusion are at the base of many diseases. Mitofusins power membrane-remodeling events via self-interaction and GTP hydrolysis. However, how exactly mitofusins mediate fusion of the outer membrane is still unclear. Structural studies enable tailored design of mitofusin variants, providing valuable tools to dissect this stepwise process. Here, we found that the two cysteines conserved between yeast and mammals are required for mitochondrial fusion, revealing two novel steps of the fusion cycle. C381 is dominantly required for the formation of the trans-tethering complex, before GTP hydrolysis. C805 allows stabilizing the Fzo1 protein and the trans-tethering complex, just prior to membrane fusion. Moreover, proteasomal inhibition rescued Fzo1 C805S levels and membrane fusion, suggesting a possible application for clinically approved drugs. Together, our study provides insights into how assembly or stability defects in mitofusins might cause mitofusin-associated diseases and uncovers potential therapeutic intervention by proteasomal inhibition.

Protein Interaction Screen on a Peptide Matrix (PrISMa).

Protein-protein interactions (PPI) are essential to understanding the cellular function and key mechanisms necessary for life. Although understanding of the interactome and proteome has exploded due to high-throughput methods in the past decade, often limitations in technical methods result in a partial understanding of all PPI. Here we present a protocol dedicated to the Protein Interaction Screen on a peptide Matrix (PrISMa). PrISMa functions as a high-throughput screen unique to targeting weak and transient interactions often missed in other PPI methods. In addition, PrISMa also excels at the mapping of interactions across linear sequences of proteins that are commonly enriched in intrinsically disordered regions (IDRs) which cover 35-40% of the mammalian proteome. This protocol aims to expand the understanding of the targeted proteins by identifying transient interactors.

A proteomics analysis of 5xFAD mouse brain regions reveals the lysosome-associated protein Arl8b as a candidate biomarker for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the intra- and extracellular accumulation of amyloid-ß (Aß) peptides. How Aß aggregates perturb the proteome in brains of patients and AD transgenic mouse models, remains largely unclear. State-of-the-art mass spectrometry (MS) methods can comprehensively detect proteomic alterations, providing relevant insights unobtainable with transcriptomics investigations. Analyses of the relationship between progressive Aß aggregation and protein abundance changes in brains of 5xFAD transgenic mice have not been reported previously. METHODS: We quantified progressive Aß aggregation in hippocampus and cortex of 5xFAD mice and controls with immunohistochemistry and membrane filter assays. Protein changes in different mouse tissues were analyzed by MS-based proteomics using label-free quantification; resulting MS data were processed using an established pipeline. Results were contrasted with existing proteomic data sets from postmortem AD patient brains. Finally, abundance changes in the candidate marker Arl8b were validated in cerebrospinal fluid (CSF) from AD patients and controls using ELISAs. RESULTS: Experiments revealed faster accumulation of Aß42 peptides in hippocampus than in cortex of 5xFAD mice, with more protein abundance changes in hippocampus, indicating that Aß42 aggregate deposition is associated with brain region-specific proteome perturbations. Generating time-resolved data sets, we defined Aß aggregate-correlated and anticorrelated proteome changes, a fraction of which was conserved in postmortem AD patient brain tissue, suggesting that proteome changes in 5xFAD mice mimic disease-relevant changes in human AD. We detected a positive correlation between Aß42 aggregate deposition in the hippocampus of 5xFAD mice and the abundance of the lysosome-associated small GTPase Arl8b, which accumulated together with axonal lysosomal membranes in close proximity of extracellular Aß plaques in 5xFAD brains. Abnormal aggregation of Arl8b was observed in human AD brain tissue. Arl8b protein levels were significantly increased in CSF of AD patients. CONCLUSIONS: We report a comprehensive biochemical and proteomic investigation of hippocampal and cortical brain tissue derived from 5xFAD transgenic mice, providing a valuable resource to the neuroscientific community. We identified Arl8b, with significant abundance changes in 5xFAD and AD patient brains. Arl8b might enable the measurement of progressive lysosome accumulation in AD patients and have clinical utility as a candidate biomarker.

Quantification of 782 Plasma Peptides by Multiplexed Targeted Proteomics.

Blood analysis is one of the foundations of clinical diagnostics. In recent years, the analysis of proteins in blood samples by mass spectrometry has taken a jump forward in terms of sensitivity and the number of identified proteins. The recent development of parallel reaction monitoring with parallel accumulation and serial fragmentation (prm-PASEF) combines ion mobility as an additional separation dimension. This increases the proteome coverage while allowing the use of shorter chromatographic gradients. To demonstrate the method's full potential, we used an isotope-labeled synthetic peptide mix of 782 peptides, derived from 579 plasma proteins, spiked into blood plasma samples with a prm-PASEF measurement allowing the quantification of 565 plasma proteins by targeted proteomics. As a less time-consuming alternative to the prm-PASEF method, we describe guided data independent acquisition (dia)-PASEF (g-dia-PASEF) and compare its application to prm-PASEF for measuring blood plasma. To demonstrate both methods' performance in clinical samples, 20 patient plasma samples from a colorectal cancer (CRC) cohort were analyzed. The analysis identified 14 differentially regulated proteins between the CRC patient and control individual plasma samples. This shows the technique's potential for the rapid and unbiased screening of blood proteins, abolishing the need for the preselection of potential biomarker proteins.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL.

Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

Cervical Fluids Are a Source of Protein Biomarkers for Early, Non-Invasive Endometrial Cancer Diagnosis.

BACKGROUND: Abnormal uterine bleeding is the main symptom of endometrial cancer (EC), but it is highly nonspecific. This represents a huge burden for women's health since all women presenting with bleeding will undergo sequential invasive tests, which are avoidable for 90-95% of those women who do not have EC. METHODS: This study aimed to evaluate the potential of cervical samples collected with five different devices as a source of protein biomarkers to diagnose EC. We evaluated the protein quantity and the proteome composition of five cervical sampling methods. RESULTS: Samples collected with a Rovers Cervex Brush® and the HC2 DNA collection device, Digene, were the most suitable samples for EC proteomic studies. Most proteins found in uterine fluids were also detected in both cervical samples. We then conducted a clinical retrospective study to assess the expression of 52 EC-related proteins in 41 patients (22 EC; 19 non-EC), using targeted proteomics. We identified SERPINH1, VIM, TAGLN, PPIA, CSE1L, and CTNNB1 as potential protein biomarkers to discriminate between EC and symptomatic non-EC women with abnormal uterine bleeding in cervical fluids (AUC > 0.8). CONCLUSIONS: This study opens an avenue for developing non-invasive protein-based EC diagnostic tests, which will improve the standard of care for gynecological patients.

An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets (MDA-MB-231) during immunological synapse formation.

Here, we present a protocol to identify and quantify phosphopeptides during the dynamic formation of an immunological synapse. We describe steps for mixing isotope-labeled immune and target cells, the stabilization of cell-to-cell conjugates by cross-linking, and their isolation by fluorescence-activated cell sorting. We detail the isolation of phosphopeptides by phosphopeptide enrichment and their subsequent measurement by mass spectrometry. Finally, we describe the analysis of the resulting data to separate cell-specific phosphopeptides using the isotope label and label-free quantification.

Disease- and sex-specific differences in patients with heart valve disease: a proteome study.

Pressure overload in patients with aortic valve stenosis and volume overload in mitral valve regurgitation trigger specific forms of cardiac remodeling; however, little is known about similarities and differences in myocardial proteome regulation. We performed proteome profiling of 75 human left ventricular myocardial biopsies (aortic stenosis = 41, mitral regurgitation = 17, and controls = 17) using high-resolution tandem mass spectrometry next to clinical and hemodynamic parameter acquisition. In patients of both disease groups, proteins related to ECM and cytoskeleton were more abundant, whereas those related to energy metabolism and proteostasis were less abundant compared with controls. In addition, disease group-specific and sex-specific differences have been observed. Male patients with aortic stenosis showed more proteins related to fibrosis and less to energy metabolism, whereas female patients showed strong reduction in proteostasis-related proteins. Clinical imaging was in line with proteomic findings, showing elevation of fibrosis in both patient groups and sex differences. Disease- and sex-specific proteomic profiles provide insight into cardiac remodeling in patients with heart valve disease and might help improve the understanding of molecular mechanisms and the development of individualized treatment strategies.

Extracellular Vesicle Secretion by Leukemia Cells In Vivo Promotes CLL Progression by Hampering Antitumor T-cell Responses.

Small extracellular vesicle (sEV, or exosome) communication among cells in the tumor microenvironment has been modeled mainly in cell culture, whereas their relevance in cancer pathogenesis and progression in vivo is less characterized. Here we investigated cancer-microenvironment interactions in vivo using mouse models of chronic lymphocytic leukemia (CLL). sEVs isolated directly from CLL tissue were enriched in specific miRNA and immune-checkpoint ligands. Distinct molecular components of tumor-derived sEVs altered CD8+ T-cell transcriptome, proteome, and metabolome, leading to decreased functions and cell exhaustion ex vivo and in vivo. Using antagomiRs and blocking antibodies, we defined specific cargo-mediated alterations on CD8+ T cells. Abrogating sEV biogenesis by Rab27a/b knockout dramatically delayed CLL pathogenesis. This phenotype was rescued by exogenous leukemic sEV or CD8+ T-cell depletion. Finally, high expression of sEV-related genes correlated with poor outcomes in CLL patients, suggesting sEV profiling as a prognostic tool. In conclusion, sEVs shape the immune microenvironment during CLL progression. SIGNIFICANCE: sEVs produced in the leukemia microenvironment impair CD8+ T-cell mediated antitumor immune response and are indispensable for leukemia progression in vivo in murine preclinical models. In addition, high expression of sEV-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients. See related commentary by Zhong and Guo, p. 5. This article is highlighted in the In This Issue feature, p. 1.

Allergic airway inflammation delays glioblastoma progression and reinvigorates systemic and local immunity in mice.

BACKGROUND: Numerous patient-based studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanisms behind this observation remain elusive. Our objective was to establish a preclinical model able to recapitulate this phenomenon and investigate the role of immunity underlying such protection. METHODS: An immunocompetent mouse model of allergic airway inflammation (AAI) was initiated before intracranial implantation of mouse GBM cells (GL261). RAG1-KO mice served to assess tumor growth in a model deficient for adaptive immunity. Tumor development was monitored by MRI. Microglia were isolated for functional analyses and RNA-sequencing. Peripheral as well as tumor-associated immune cells were characterized by flow cytometry. The impact of allergy-related microglial genes on patient survival was analyzed by Cox regression using publicly available datasets. RESULTS: We found that allergy establishment in mice delayed tumor engraftment in the brain and reduced tumor growth resulting in increased mouse survival. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state, uncovering a microglia gene signature, which correlated with limited local immunosuppression in glioma patients. AAI increased effector memory T-cells in the circulation as well as tumor-infiltrating CD4+ T-cells. The survival benefit conferred by AAI was lost in mice devoid of adaptive immunity. CONCLUSION: Our results demonstrate that AAI limits both tumor take and progression in mice, providing a preclinical model to study the impact of allergy on GBM susceptibility and prognosis, respectively. We identify a potentiation of local and adaptive systemic immunity, suggesting a reciprocal crosstalk that orchestrates allergy-induced immune protection against GBM.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

Pan-claudin family interactome analysis reveals shared and specific interactions.

Claudins are a family of transmembrane proteins expressed in epithelial tissues and are the major components of tight junctions (TJs), which define barrier properties in epithelia and maintain cell polarity. How claudins regulate the formation of TJs and which functions they exert outside of them is not entirely understood. Although the long and unstructured C-terminal tail is essential for regulation, it is unclear how it is involved in these functions beyond interacting with TJ-associated proteins such as TJ protein ZO-1 (TJP1). Here, we present an interactome study of the pan-claudin family in Madin-Darby canine kidney (MDCK)-C7 cells by combining two complementary mass spectrometry-based pull-down techniques creating an interaction landscape of the entire claudin family. The interaction partners of the claudins' C termini reveal their possible implications in localized biological processes in epithelial cells and their regulation by post-translational modifications (PTMs).

Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae: Protein kinase Tda1 is essential for Hxk1 and Hxk2 serine 15 phosphorylation.

Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. To additionally address possible consequences of glucose repression/derepression, both were grown at 2% and 0.1% (w/v) glucose. A total of eight protein spots exhibiting a minimum twofold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. Among the spot identities are-besides the expected Hxk2-two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. Thirty-six glucose-concentration-dependent protein spots were identified. A simple method to improve spot quantification, approximating spots as rotationally symmetric solids, is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth under high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts.

Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further colocalized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues.