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The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological activities. These GPI-APs occupy distinct cellular functions such as enzymes, receptors, and adhesion molecules, and they are implicated in several vital cellular processes. Thus, unraveling the mechanisms and regulators of their membrane organization is essential. In polarized epithelial cells, GPI-APs are enriched at the apical surface, where they form small cholesterol-independent homoclusters and larger heteroclusters accommodating multiple GPI-AP species, all confined within areas of approximately 65-70 nm in diameter. Notably, GPI-AP homoclustering occurs in the Golgi apparatus through a cholesterol- and calcium-dependent mechanism that drives their apical sorting. Despite the critical role of Golgi GPI-AP clustering in their cell surface organization and the importance of cholesterol in heterocluster formation, the regulatory mechanisms governing GPI-AP surface organization, particularly in the context of epithelial polarity, remain elusive. Given that the actin cytoskeleton undergoes substantial remodeling during polarity establishment, this study explores whether the actin cytoskeleton regulates the spatiotemporal apical organization of GPI-APs in MDCK cells. Utilizing various imaging techniques (number and brightness, FRET/FLIM, and dSTORM coupled to pair correlation analysis), we demonstrate that the apical organization of GPI-APs, at different scales, does not rely on the actin cytoskeleton, unlike in fibroblastic cells. Interestingly, calcium chelation disrupts the organization of GPI-APs at the apical surface by impairing Golgi GPI-AP clustering, emphasizing the existence of an interplay among Golgi clustering, apical sorting, and surface organization in epithelial cells. In summary, our findings unveil distinct mechanisms regulating the organization of GPI-APs in cell types of different origins, plausibly allowing them to adapt to different external signals and different cellular environments in order to achieve specialized functions.

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KEY MESSAGE: GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule-polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.

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Pollen tube integrity is required for achieving double fertilization in angiosperms. The rapid alkalinization factor4/19-ANXUR1/2-Buddha's paper seal 1/2 (RALF4/19-ANX1/2-BUPS1/2)-complex-mediated signaling pathway is critical to maintain pollen tube integrity, but the underlying mechanisms regulating the polar localization and distribution of these complex members at the pollen tube tip remain unclear. Here, we find that COBRA-like protein 11 (COBL11) loss-of-function mutants display a low pollen germination ratio, premature pollen tube burst, and seed abortion in Arabidopsis. COBL11 could interact with RALF4/19, ANX1/2, and BUPS1/2, and COBL11 functional deficiency could result in the disrupted distribution of RALF4 and ANX1, altered cell wall composition, and decreased levels of reactive oxygen species in pollen tubes. In conclusion, COBL11 is a regulator of pollen tube integrity during polar growth, which is conducted by a direct interaction that ensures the correct localization and polar distribution of RALF4 and ANX1 at the pollen tube tip.

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Although extracellular vesicles (EVs) were discovered over 40 years ago, there has been a resurgence of interest in secreted vesicles and their attendant cargo as novel modes of intracellular communication. In addition to vesicles, two amembranous nanoparticles, exomeres and supermeres, have been isolated and characterized recently. In this rapidly expanding field, it has been challenging to assign cargo and specific functions to a particular carrier. Refinement of isolation methods, well-controlled studies, and guidelines detailed by Minimal Information for Studies of Extracellular Vesicles (MISEV) are being employed to "bring order to chaos." In this review, we will briefly summarize three types of extracellular carriers - small EVs (sEVs), exomeres, and supermeres - in the context of colorectal cancer (CRC). We found that a number of GPI-anchored proteins (GPI-APs) are overexpressed in CRC, are enriched in exosomes (a distinct subset of sEVs), and can be detected in exomeres and supermeres. This affords the opportunity to elaborate on GPI-AP biogenesis, modifications, and trafficking using DPEP1, a GPI-AP upregulated in CRC, as a prime example. We have cataloged the GPI-anchored proteins secreted in CRC and will highlight features of select CRC-associated GPI-anchored proteins we have detected. Finally, we will discuss the remaining challenges and future opportunities in studying these secreted GPI-APs in CRC.

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In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

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Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of the plasma membrane (PM) bilayer by covalent linkage to a typical glycolipid and expressed in all eukaryotic organisms so far studied. Lipolytic release from PMs into extracellular compartments and intercellular transfer are regarded as the main (patho)physiological roles exerted by GPI-APs. The intercellular transfer of GPI-APs relies on the complete GPI anchor and is mediated by extracellular vesicles such as microvesicles and exosomes and lipid-free homo- or heteromeric aggregates, and lipoprotein-like particles such as prostasomes and surfactant-like particles, or lipid-containing micelle-like complexes. In mammalian organisms, non-vesicular transfer is controlled by the distance between donor and acceptor cells/tissues; intrinsic conditions such as age, metabolic state, and stress; extrinsic factors such as GPI-binding proteins; hormones such as insulin; and drugs such as anti-diabetic sulfonylureas. It proceeds either "directly" upon close neighborhood or contact of donor and acceptor cells or "indirectly" as a consequence of the induced lipolytic release of GPI-APs from PMs. Those displace from the serum GPI-binding proteins GPI-APs, which have retained the complete anchor, and become assembled in aggregates or micelle-like complexes. Importantly, intercellular transfer of GPI-APs has been shown to induce specific phenotypes such as stimulation of lipid and glycogen synthesis, in cultured human adipocytes, blood cells, and induced pluripotent stem cells. As a consequence, intercellular transfer of GPI-APs should be regarded as non-genetic inheritance of (acquired) features between somatic cells which is based on the biogenesis and transmission of matter such as GPI-APs and "membrane landscapes", rather than the replication and transmission of information such as DNA. Its operation in mammalian organisms remains to be clarified.

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Endoplasmic reticulum (ER) stress and unfolded protein response are cells' survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a pro-PrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPI-anchored PrP to pro-PrP via a conserved ATF6-miRNA449c-5p-PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 3'-UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6-miR449c-5p-PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression.

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Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript).

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Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are anchored at the outer leaflet of eukaryotic plasma membranes (PMs) only by carboxy-terminal covalently coupled GPI. GPI-APs are known to be released from the surface of donor cells in response to insulin and antidiabetic sulfonylureas (SUs) by lipolytic cleavage of the GPI or upon metabolic derangement as full-length GPI-APs with the complete GPI attached. Full-length GPI-APs become removed from extracellular compartments by binding to serum proteins, such as GPI-specific phospholipase D (GPLD1), or insertion into the PMs of acceptor cells. Here, the interplay between the lipolytic release and intercellular transfer of GPI-APs and its potential functional impact was studied using transwell co-culture with human adipocytes as insulin-/SU-responsive donor cells and GPI-deficient erythroleukemia as acceptor cells (ELCs). Measurement of the transfer as the expression of full-length GPI-APs at the ELC PMs by their microfluidic chip-based sensing with GPI-binding α-toxin and GPI-APs antibodies and of the ELC anabolic state as glycogen synthesis upon incubation with insulin, SUs and serum yielded the following results: (i) Loss of GPI-APs from the PM upon termination of their transfer and decline of glycogen synthesis in ELCs, as well as prolongation of the PM expression of transferred GPI-APs upon inhibition of their endocytosis and upregulated glycogen synthesis follow similar time courses. (ii) Insulin and SUs inhibit both GPI-AP transfer and glycogen synthesis upregulation in a concentration-dependent fashion, with the efficacies of the SUs increasing with their blood glucose-lowering activity. (iii) Serum from rats eliminates insulin- and SU-inhibition of both GPI-APs' transfer and glycogen synthesis in a volume-dependent fashion, with the potency increasing with their metabolic derangement. (iv) In rat serum, full-length GPI-APs bind to proteins, among them (inhibited) GPLD1, with the efficacy increasing with the metabolic derangement. (v) GPI-APs are displaced from serum proteins by synthetic phosphoinositolglycans and then transferred to ELCs with accompanying stimulation of glycogen synthesis, each with efficacies increasing with their structural similarity to the GPI glycan core. Thus, both insulin and SUs either block or foster transfer when serum proteins are depleted of or loaded with full-length GPI-APs, respectively, i.e., in the normal or metabolically deranged state. The transfer of the anabolic state from somatic to blood cells over long distance and its "indirect" complex control by insulin, SUs and serum proteins support the (patho)physiological relevance of the intercellular transfer of GPI-APs.

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Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. Cerebellar lipid rafts are cell surface ganglioside microdomains for the attachment of GPI-anchored neural adhesion molecules and downstream signaling molecules such as Src-family kinases and heterotrimeric G proteins. In this review, we summarize our recent findings on signaling in ganglioside GD3 rafts of cerebellar granule cells and several findings by other groups on the roles of lipid rafts in the cerebellum. TAG-1, of the contactin group of immunoglobulin superfamily cell adhesion molecules, is a phosphacan receptor. Phosphacan regulates the radial migration signaling of cerebellar granule cells, via Src-family kinase Lyn, by binding to TAG-1 on ganglioside GD3 rafts. Chemokine SDF-1α, which induces the tangential migration of cerebellar granule cells, causes heterotrimeric G protein Goα translocation to GD3 rafts. Furthermore, the functional roles of cerebellar raft-binding proteins including cell adhesion molecule L1, heterotrimeric G protein Gsα, and L-type voltage-dependent calcium channels are discussed.

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This year celebrates the 50th anniversary of the Singer-Nicolson fluid mosaic model for biological membranes. The next level of sophistication we have achieved for understanding plasma membrane (PM) structures, dynamics, and functions during these 50 years includes the PM interactions with cortical actin filaments and the partial demixing of membrane constituent molecules in the PM, particularly raft domains. Here, first, we summarize our current knowledge of these two structures and emphasize that they are interrelated. Second, we review the structure, molecular dynamics, and function of raft domains, with main focuses on raftophilic glycosylphosphatidylinositol-anchored proteins (GPI-APs) and their signal transduction mechanisms. We pay special attention to the results obtained by single-molecule imaging techniques and other advanced microscopy methods. We also clarify the limitations of present optical microscopy methods for visualizing raft domains, but emphasize that single-molecule imaging techniques can "detect" raft domains associated with molecules of interest in the PM.

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The protozoa Leishmania donovani causes visceral leishmaniasis (kala-azar), the third most common vector-borne disease. The visceral organs, particularly the spleen, liver, and bone marrow, are affected by the disease. The lack of effective treatment regimens makes curing and eradicating the disease difficult. The availability of complete L. donovani genome/proteome data allows for the development of specific and efficient vaccine candidates using the reverse vaccinology method, while utilizing the unique sequential and structural features of potential antigenic proteins to induce protective T cell and B cell responses. Such shortlisted candidates may then be tested quickly for their efficacy in the laboratory and later in clinical settings. These antigens will also be useful for designing antigen-based next-generation sero-diagnostic assays. L. donovani's cell surface-associated proteins and secretory proteins are among the first interacting entities to be exposed to the host immune machinery. As a result, potential antigenic epitope peptides derived from these proteins could serve as competent vaccine components. We used a stepwise filtering-based in silico approach to identify the entire surface-associated and secretory proteome of L. donovani, which may provide rationally selected most exposed antigenic proteins. Our study identified 12 glycosylphosphatidylinositol-anchored proteins, 45 transmembrane helix-containing proteins, and 73 secretory proteins as potent antigens unique to L. donovani. In addition, we used immunoinformatics to identify B and T cell epitopes in them. Out of the shortlisted surface-associated and secretory proteome, 66 protein targets were found to have the most potential overlapping B cell and T cell epitopes (linear and conformational; MHC class I and MHC class II).

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Glycosylphosphatidylinositol-anchored proteins (GPI-APs), which are anchored at the outer leaflet of plasma membranes (PM) only by a carboxy-terminal GPI glycolipid, are known to fulfill multiple enzymic and receptor functions at the cell surface. Previous studies revealed that full-length GPI-APs with the complete GPI anchor attached can be released from and inserted into PMs in vitro. Moreover, full-length GPI-APs were recovered from serum, dependent on the age and metabolic state of rats and humans. Here, the possibility of intercellular control of metabolism by the intercellular transfer of GPI-APs was studied. Mutant K562 erythroleukemia (EL) cells, mannosamine-treated human adipocytes and methyl-ß-cyclodextrin-treated rat adipocytes as acceptor cells for GPI-APs, based on their impaired PM expression of GPI-APs, were incubated with full-length GPI-APs, prepared from rat adipocytes and embedded in micelle-like complexes, or with EL cells and human adipocytes with normal expression of GPI-APs as donor cells in transwell co-cultures. Increases in the amounts of full-length GPI-APs at the PM of acceptor cells as a measure of their transfer was assayed by chip-based sensing. Both experimental setups supported both the transfer and upregulation of glycogen (EL cells) and lipid (adipocytes) synthesis. These were all diminished by serum, serum GPI-specific phospholipase D, albumin, active bacterial PI-specific phospholipase C or depletion of total GPI-APs from the culture medium. Serum inhibition of both transfer and glycogen/lipid synthesis was counteracted by synthetic phosphoinositolglycans (PIGs), which closely resemble the structure of the GPI glycan core and caused dissociation of GPI-APs from serum proteins. Finally, large, heavily lipid-loaded donor and small, slightly lipid-loaded acceptor adipocytes were most effective in stimulating transfer and lipid synthesis. In conclusion, full-length GPI-APs can be transferred between adipocytes or between blood cells as well as between these cell types. Transfer and the resulting stimulation of lipid and glycogen synthesis, respectively, are downregulated by serum proteins and upregulated by PIGs. These findings argue for the (patho)physiological relevance of the intercellular transfer of GPI-APs in general and its role in the paracrine vs. endocrine (dys)regulation of metabolism, in particular. Moreover, they raise the possibility of the use of full-length GPI-APs as therapeutics for metabolic diseases.

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the viability of upper and lower motor neurons. Current options for treatment are limited, necessitating deeper understanding of the mechanisms underlying ALS pathogenesis. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that acts on the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane. GDE2 is required for the survival of spinal motor neurons but whether GDE2 neuroprotective activity is disrupted in ALS is not known. We utilized a combination of mouse models and patient post-mortem samples to evaluate GDE2 functionality in ALS. Haplogenetic reduction of GDE2 exacerbated motor neuron degeneration and loss in SOD1G93A mice but not in control SOD1WT transgenic animals, indicating that GDE2 neuroprotective function is diminished in the context of SOD1G93A. In tissue samples from patients with ALS, total levels of GDE2 protein were equivalent to healthy controls; however, membrane levels of GDE2 were substantially reduced. Indeed, GDE2 was found to aberrantly accumulate in intracellular compartments of ALS motor cortex, consistent with a disruption of GDE2 function at the cell surface. Supporting the impairment of GDE2 activity in ALS, tandem-mass-tag mass spectrometry revealed a pronounced reduction of GPI-anchored proteins released into the CSF of patients with ALS compared with control patients. Taken together, this study provides cellular and biochemical evidence that GDE2 distribution and activity is disrupted in ALS, supporting the notion that the failure of GDE2-dependent neuroprotective pathways contributes to neurodegeneration and motor neuron loss in disease. These observations highlight the dysregulation of GPI-anchored protein pathways as candidate mediators of disease onset and progression and accordingly, provide new insight into the mechanisms underlying ALS pathogenesis.

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It is becoming increasingly obvious that glycophosphatidylinositol (GPI)-anchored proteins (GAPs) play a prominent role in fungi, a full understanding of GAPs is however lacking especially for the human opportunistic fungus Cryptococcus neoformans. Using online GPI prediction tools, GAPs were identified and subsequently a mutant library for these GAP-encoding genes was developed and a publicly available knock out (KO) mutant library was used. In total, 41 overexpression and 34 KO mutants, representing 47 unique genes, were analyzed. From the analysis of the two libraries, two main gene candidates, a mannoprotein 88 (MP88) (CNAG_00776) and an uncharacterized protein (CNAG_00137) were further investigated by constructing additional independent mutant strains. The CNAG_00776 mutant showed an impaired growth upon plasma membrane stress and significant decreased phagocytosis. The CNAG_00137 mutant showed impaired growth during cell wall stress or increased temperature and significant decreased phagocytosis. By performing a large genetic screen of GAPs in the genome of the human fungal pathogen C. neoformans, we identified two candidate GAP genes involved in C. neoformans/host interaction and stress response. Further research into these two genes could potentially result in new targets for antfungals, treatment strategies or vaccines to manage C. neoformans disease.

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Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1/PGAP3 like genes (AtPGAP3A and AtPGAP3B). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus.

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BACKGROUND: Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial. RESULTS: We have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a > 34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains. CONCLUSION: Our results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of action of Bt toxins, which can compensate for the absence of individual receptors, and are consistent with an interplay among diverse midgut receptors in the toxins' mechanism of action in a super pest.

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Diversity in arbuscular mycorrhizal fungi (AMF) contributes to biodiversity and resilience in natural environments and healthy agricultural systems. Functional complementarity exists among species of AMF in symbiosis with their plant hosts, but the molecular basis of this is not known. We hypothesise this is in part due to the difficulties that current sequence assembly methodologies have assembling sequences for intrinsically disordered proteins (IDPs) due to their low sequence complexity. IDPs are potential candidates for functional complementarity because they often exist as extended (non-globular) proteins providing additional amino acids for molecular interactions. Rhizophagus irregularis arabinogalactan-protein-like proteins (AGLs) are small secreted IDPs with no known orthologues in AMF or other fungi. We developed a targeted bioinformatics approach to identify highly variable AGLs/IDPs in RNA-sequence datasets. The approach includes a modified multiple k-mer assembly approach (Oases) to identify candidate sequences, followed by targeted sequence capture and assembly (mirabait-mira). All AMF species analysed, including the ancestral family Paraglomeraceae, have small families of proteins rich in disorder promoting amino acids such as proline and glycine, or glycine and asparagine. Glycine- and asparagine-rich proteins also were found in Geosiphon pyriformis (an obligate symbiont of a cyanobacterium), from the same subphylum (Glomeromycotina) as AMF. The sequence diversity of AGLs likely translates to functional diversity, based on predicted physical properties of tandem repeats (elastic, amyloid, or interchangeable) and their broad pI ranges. We envisage that AGLs/IDPs could contribute to functional complementarity in AMF through processes such as self-recognition, retention of nutrients, soil stability, and water movement.

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Complexes of p24 proteins act as cargo receptors for the transport of COPII vesicles from the endoplasmic reticulum (ER). The major cargos of p24 complexes are hydrophilic proteins tethered to the ER membrane via a covalently attached glycosylphosphatidylinositol (GPI) or fatty acid. Each p24 complex is known to contain members from all four p24 subfamilies (p24α, p24ß, p24γ and p24δ). However, it remains unclear how the cargo specificities of p24 complexes are influenced by member stoichiometry. Here, we report the subunit compositions of mammalian p24 complexes involved in the transport of GPI-anchored proteins and Wnt1. We show that at least one p24α is required for the formation of p24 complexes and that a p24 complex consisting of p24α2, p24ß1, p24γ2 and p24δ1 is required for the efficient transport of GPI-anchored proteins. On the other hand, a p24 complex containing p24α2, p24α3, p24ß1, p24γ and p24δ1 is involved in the transport of Wnt1. Further, interactions between p24α2 and p24α3 are critical for Wnt1 transport. Thus, p24α and p24γ subfamily members are important for cargo selectivity. Lastly, our data fit with an octamer, rather than a tetramer, model of p24 complexes, where each complex consists of two proteins from each p24 subfamily.

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Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single ß-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal 'pre-orientation' of the LU domain for the receptor interaction.

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