Lipid Transfer Protein Family Research Articles

Bridge-like lipid transfer protein family member 2 suppresses ciliogenesis.

Bridge-like lipid transfer protein family member 2 (BLTP2) is an evolutionary conserved protein with unknown function(s). The absence of BLTP2 in Drosophila melanogaster results in impaired cellular secretion and larval death, while in mice (Mus musculus), it causes preweaning lethality. Structural predictions propose that BLTP2 belongs to the repeating β-groove domain-containing (also called the VPS13) protein family, forming a long tube with a hydrophobic core, suggesting that it operates as a lipid transfer protein (LTP). We establish BLTP2 as a negative regulator of ciliogenesis in RPE-1 cells based on a strong genetic interaction with WDR44, a gene that also suppresses ciliogenesis. Like WDR44, BLTP2 localizes to membrane contact sites involving the endoplasmic reticulum and the tubular endosome network in HeLa cells and that BLTP2 depletion enhanced ciliogenesis in RPE-1 cells grown in serum-containing medium, a condition where ciliogenesis is normally suppressed. This study establishes human BLTP2 as a putative LTP acting between tubular endosomes and ER that regulates primary cilium biogenesis.

Promiscuous Artemisia annua glycosylphosphatidylinositol-anchored lipid transfer protein 1 (AaLTPG1) promotes terpenoids accumulation in planta

LTPG, encoded by a glycosylphosphatidylinositol-anchored lipid transfer protein family gene, plays a role in exporting fatty acids to the plant surface through the hydrophilic cell wall layer. While soluble extracellular LTPs are known to function in terpenoid accumulation in trichomes, whether membrane-integrated LTPG influences terpenoid accumulation and its underlying mechanisms remains poorly understood. In this study, we report an Artemisia annua LTPG1, which is strongly expressed in floral tissues and positively correlated with three artemisinin biosynthetic genes. The recombinant AaLTPG1 displayed promiscuous binding activity with fatty acids and artemisinin precursors in vitro, resulting in an increase in terpenoids and lipids contents in both AaLTPG1 transgenic tobacco without altering trichome initiation, development, or secretion. Transcriptome sequencing further revealed that ethylene responsive genes were repressed in AaLTPG1-overexpressing tobacco plants. Consistently, artemisinin and its precursors were found to increase transiently in A. annua leaves, and the expression level of ethylene-responsive factors (ERFs) was down-regulated in A. annua. Collectively, this work illustrates the positive regulatory role of LTPG in terpenoids and lipids accumulation by exporting hydrophobic metabolites and repressing ethylene signaling, making AaLTPG1 a potential ideal target for metabolic engineering to improve artemisinin accumulation.

RBG Motif Bridge-Like Lipid Transport Proteins: Structure, Functions, and Open Questions.

The life of eukaryotic cells requires the transport of lipids between membranes, which are separated by the aqueous environment of the cytosol. Vesicle-mediated traffic along the secretory and endocytic pathways and lipid transfer proteins (LTPs) cooperate in this transport. Until recently, known LTPs were shown to carry one or a few lipids at a time and were thought to mediate transport by shuttle-like mechanisms. Over the last few years, a new family of LTPs has been discovered that is defined by a repeating β-groove (RBG) rod-like structure with a hydrophobic channel running along their entire length. This structure and the localization of these proteins at membrane contact sites suggest a bridge-like mechanism of lipid transport. Mutations in some of these proteins result in neurodegenerative and developmental disorders. Here we review the known properties and well-established or putative physiological roles of these proteins, and we highlight the many questions that remain open about their functions.

Diversity of Cationic Antimicrobial Peptides in Black Cumin (Nigella sativa L.) Seeds.

Black cumin (Nigella sativa L.) is known to possess a wide variety of antimicrobial peptides belonging to different structural families. Three novel antimicrobial peptides have been isolated from black cumin seeds. Two of them were attributed as members of the non-specific lipid transfer proteins family, and one as a defensin. We have made an attempt of using the proteomic approach for novel antimicrobial peptides search in N. sativa seeds as well. The use of a well-established approach that includes extraction and fractionation stages remains relevant even in the case of novel peptides search because of the lacking N. sativa genome data. Novel peptides demonstrate a spectrum of antimicrobial activity against plant pathogenic organisms that may cause economically important crop diseases. These results obtained allow considering these molecules as candidates to be applied in "next-generation" biopesticides development for agricultural use.

Identification of potato Lipid transfer protein gene family and expression verification of drought genes StLTP1 and StLTP7

Lipid transfer proteins (LTPs) are widely distributed in plants and play an important role in the response to stress. Potato (Solanum tuberosum L.) is sensitive to a lack of water, and drought stress is one of the limiting factors for its yield. Therefore, mining candidate functional genes for drought stress and creating new types of potato germplasm for drought resistance is an effective way to solve this problem. There are few reports on the LTP family in potato. In this study, 39 members of the potato LTP family were identified. They were located on seven chromosomes, and the amino acid sequences encoded ranged from 101 to 345 aa. All 39 family members contained introns and had exons that ranged from one to four. Conserved motif analysis of potato LTP transcription factors showed that 34 transcription factors contained Motif 2 and Motif 4, suggesting that they were conserved motifs of potato LTP. Compared with the LTP genes of homologous crops, the potato and tomato (Solanum lycopersicum L.) LTPs were the mostly closely related. The StLTP1 and StLTP7 genes were screened by quantitative reverse transcription PCR combined with potato transcriptome data to study their expression in tissues and the characteristics of their responses to drought stress. The results showed that StLTP1 and StLTP7 were upregulated in the roots, stems, and leaves after PEG 6000 stress. Taken together, our study provides comprehensive information on the potato LTP family that will help to develop a framework for further functional studies.

ORP9 and ORP10 form a heterocomplex to transfer phosphatidylinositol 4-phosphate at ER-TGN contact sites.

Oxysterol-binding protein (OSBP) and its related proteins (ORPs) are a family of lipid transfer proteins (LTPs) that mediate non-vesicular lipid transport. ORP9 and ORP10, members of the OSBP/ORPs family, are located at the endoplasmic reticulum (ER)-trans-Golgi network (TGN) membrane contact sites (MCSs). It remained unclear how they mediate lipid transport. In this work, we discovered that ORP9 and ORP10 form a binary complex through intermolecular coiled-coil (CC) domain-CC domain interaction. The PH domains of ORP9 and ORP10 specially interact with phosphatidylinositol 4-phosphate (PI4P), mediating the TGN targeting. The ORP9-ORP10 complex plays a critical role in regulating PI4P levels at the TGN. Using in vitro reconstitution assays, we observed that while full-length ORP9 efficiently transferred PI4P between two apposed membranes, the lipid transfer kinetics was further accelerated by ORP10. Interestingly, our data showed that the PH domains of ORP9 and ORP10 participate in membrane tethering simultaneously, whereas ORDs of both ORP9 and ORP10 are required for lipid transport. Furthermore, our data showed that the depletion of ORP9 and ORP10 led to increased vesicle transport to the plasma membrane (PM). These findings demonstrate that ORP9 and ORP10 form a binary complex through the CC domains, maintaining PI4P homeostasis at ER-TGN MCSs and regulating vesicle trafficking.

Herbivory-inducible lipid-transfer proteins (LTPs) of Cicer arietinum as potential human allergens

Lipid-transfer proteins (LTPs) are lipid-binding small proteins, ubiquitously distributed amongst plant kingdom. Apart from their involvement in plant defense, it has also been discovered that they induce allergic reactions in humans. A plethora of LTPs have been identified in vegetables, fruits, pollens, nuts, and latex, among which Pru p 3, a LTP allergen from peach fruit, is extensively studied and exhibits cross-reactivity with potential allergens from different species. In Cicer arietinum, a family of LTPs (CaLTPs) has been identified and their importance in plant defense during Helicoverpa armigera-infestation has been recognized. However, the determination of the allergenicity potential of CaLTPs has not been attempted. In this study, we aim to decipher the allergenicity potential of defense-related CaLTPs. The allergenicity potential prediction, and identification of B-cell epitope binding regions showed that the CaLTPs had conserved domains and B-cell epitopes in the same regions as Prup3 (a marker allergen for LTPs). Using molecular docking and simulations, we observed that the CaLTPs successfully interacted with the Immunoglobin E(IgE)with docking energies ranging from −315.5 to −268.4 and the structures were stabilized within 10 ns of simulation. Through this study, we intend to embellish our present knowledge and understanding of the sensitization and allergenicity potential of CaLTPs. Communicated by Ramaswamy H. Sarma

A Systematic Investigation of Lipid Transfer Proteins Involved in Male Fertility and Other Biological Processes in Maize

Plant lipid transfer proteins (LTPs) play essential roles in various biological processes, including anther and pollen development, vegetative organ development, seed development and germination, and stress response, but the research progress varies greatly among Arabidopsis, rice and maize. Here, we presented a preliminary introduction and characterization of the whole 65 LTP genes in maize, and performed a phylogenetic tree and gene ontology analysis of the LTP family members in maize. We compared the research progresses of the reported LTP genes involved in male fertility and other biological processes in Arabidopsis and rice, and thus provided some implications for their maize orthologs, which will provide useful clues for the investigation of LTP transporters in maize. We predicted the functions of LTP genes based on bioinformatic analyses of their spatiotemporal expression patterns by using RNA-seq and qRT-PCR assays. Finally, we discussed the advances and challenges in substrate identification of plant LTPs, and presented the future research directions of LTPs in plants. This study provides a basic framework for functional research and the potential application of LTPs in multiple plants, especially for male sterility research and application in maize.

Phylogenetic and Structural Analyses of VPS13 Proteins in Archaeplastida Reveal Their Complex Evolutionary History in Viridiplantae.

VPS13 is a lipid transfer protein family conserved among Eukaryotes and playing roles in fundamental processes involving vesicular transport and membrane expansion including autophagy and organelle biogenesis. VPS13 folds into a long hydrophobic tunnel, allowing lipid transport, decorated by distinct domains involved in protein localization and regulation. Whereas VPS13 organization and function have been extensively studied in yeast and mammals, information in organisms originating from primary endosymbiosis is scarce. In the higher plant Arabidopsis thaliana, four paralogs, AtVPS13S, X, M1, and M2, were identified, AtVPS13S playing a role in the regulation of root growth, cell patterning, and reproduction. In this work, we performed phylogenetic, as well as domain and structural modeling of VPS13 proteins in Archaeplastida in order to understand their general organization and evolutionary history. We confirmed the presence of human VPS13B orthologues in some phyla and described two new VPS13 families presenting a particular domain arrangement: VPS13R in Rhodophytes and VPS13Y in Chlorophytes and Streptophytes. By focusing on Viridiplantae, we were able to draw the evolutionary history of these proteins made by multiple gene gains and duplications as well as domain rearrangements. We showed that some Chlorophytes have only three (AtVPS13M, S, Y) whereas some Charophytes have up to six VPS13 paralogs (AtVPS13M1, M2, S, Y, X, B). We also highlighted specific structural features of VPS13M and X paralogs. This study reveals the complex evolution of VPS13 family and opens important perspectives for their functional characterization in photosynthetic organisms.

Systematic and functional analysis of non-specific lipid transfer protein family genes in sugarcane under Xanthomonas albilineans infection and salicylic acid treatment.

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins that play a significant regulatory role in a wide range of physiological processes. To date, no genome-wide survey and expression analysis of this gene family in sugarcane has been performed. In this study we identified the nsLTP gene family in Saccharum spontaneum and carried out expression profiling of nsLTPs in two sugarcane cultivars (Saccharum spp.) that have different resistance to leaf scald caused by Xanthomonas albilineans (Xa) infection. The effect of stress related to exogenous salicylic acid (SA) treatment was also examined. At a genome-wide level, S. spontaneum AP85-441 had 71 SsnsLTP genes including 66 alleles. Tandem (9 gene pairs) and segmental (36 gene pairs) duplication events contributed to SsnsLTP gene family expansion. Five SsnsLTP proteins were predicted to interact with five other proteins. Expression of ShnsLTPI.8/10/Gb.1 genes was significantly upregulated in LCP85-384 (resistant cultivar), but downregulated in ROC20 (susceptible cultivar), suggesting that these genes play a positive regulatory role in response of sugarcane to Xa infection. Conversely, ShnsLTPGa.4/Ge.3 appears to act as a negative regulator in response Xa infection. The majority (16/17) of tested genes were positively induced in LCP85-384 72 h after SA treatment. In both cultivars, but particularly in LCP85-384, ShnsLTPIV.3/VIII.1 genes were upregulated at all time-points, suggesting that the two genes might act as positive regulators under SA stress. Meanwhile, both cultivars showed downregulated ShnsLTPGb.1 gene expression, indicating its potential negative role in SA treatment responses. Notably, the ShnsLTPGb.1 gene had contrasting effects, with positive regulation of gene expression in response to Xa infection and negative regulation induced by SA stress. Together, our results provide valuable information for elucidating the function of ShnsLTP family members under two stressors and identified novel gene sources for development of sugarcane that are tolerant of environmental stimuli.

Poplar glycosylphosphatidylinositol-anchored lipid transfer proteins respond to osmotic stress by regulating fatty acid biosynthesis

Non-specific lipid transfer proteins (LTPs) comprise various protein families in plant responses to biotic and abiotic stresses. LTPGs encoding a glycosylphosphatidylinositol (GPI)-anchored lipid transfer protein are a large subgroup within the LTP family. We identified 36 PtLTPG genes in the poplar genome, divided into four subgroups based on phylogenetic analysis. A total of 29 PtLTPG genes were amplified from the poplar genome. PtLTPG1, 2, and 31 were localized to the plasma membrane and PtLTPG9 to the nucleus, suggesting functional dissimilarity. The PtLTPG genes were expressed in all poplar tissues tested, but the expression levels of PtLTPG7 and 11 were low. Most PtLTPG and FA-related genes were induced by abiotic stresses, and the expression patterns of PtLTPGs were associated with those of FA-related genes. The FA composition, including the ratio of linolenic acid to linoleic acid, was altered by osmotic stress. Taken together, our results show that PtLTPGs and FA-related genes mediate tolerance to osmotic stress by modulating the FA composition in poplar.

The lipid transporter proteins ORP5 and ORP8 in Ca2+signaling

Oxysterol-binding protein (OSBP)-related proteins (ORPs), a family of lipid transfer proteins (LTPs), mediate the exchange of the lipids PI(4)P and phosphatidylserine (PtdSer) between the plasma membrane (PM) and the endoplasmic reticulum (ER). ORP5 and ORP8 are located and function at membrane contact sites (MCSs), including the ER/PM junctions. MCSs between the ER and PM are essential platform for Ca2+ signaling complexes and within them the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1.

Reconstitution and biochemical studies of extended synaptotagmin-mediated lipid transport.

Extended synaptotagmins (E-Syts) are a family of lipid transfer proteins (LTPs) located at the endoplasmic reticulum (ER)-plasma membrane (PM) contact sites in eukaryotic cells. They possess a conserved synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and two to five C2 domains. While the membrane tethering function of E-Syts has been well studied in diverse species, recent studies revealed that the mammalian E-Syt1 and its yeast homolog tricalbin 3 (Tcb3) could transport lipids between the opposed membrane. Mechanical studies suggested SYT1 transfers lipids fundamentally through the SMP domain, but the lipid transport requires the regulation of C2 domain-mediated membrane tethering. In addition, both E-Syt1 and Tcb3 are Ca2+-modulated LTPs, which sense and interact with Ca2+ through the C2 domains. This chapter describes the in vitro reconstitution and biochemical assays for studying the functions and mechanisms of E-Syts, by expressing and purifying recombinant proteins, preparing reconstitution systems, and developing assays for membrane tethering and lipid transport.

VPS13A and VPS13C Influence Lipid Droplet Abundance.

Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinson’s disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) – lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate two lipid transfer proteins, VPS13A and VPS13C, in LD regulation.

Functions of Oxysterol-Binding Proteins at Membrane Contact Sites and Their Control by Phosphoinositide Metabolism.

Lipids must be correctly transported within the cell to the right place at the right time in order to be fully functional. Non-vesicular lipid transport is mediated by so-called lipid transfer proteins (LTPs), which contain a hydrophobic cavity that sequesters lipid molecules. Oxysterol-binding protein (OSBP)-related proteins (ORPs) are a family of LTPs known to harbor lipid ligands, such as cholesterol and phospholipids. ORPs act as a sensor or transporter of those lipid ligands at membrane contact sites (MCSs) where two different cellular membranes are closely apposed. In particular, a characteristic functional property of ORPs is their role as a lipid exchanger. ORPs mediate counter-directional transport of two different lipid ligands at MCSs. Several, but not all, ORPs transport their lipid ligand from the endoplasmic reticulum (ER) in exchange for phosphatidylinositol 4-phosphate (PI4P), the other ligand, on apposed membranes. This ORP-mediated lipid “countertransport” is driven by the concentration gradient of PI4P between membranes, which is generated by its kinases and phosphatases. In this review, we will discuss how ORP function is tightly coupled to metabolism of phosphoinositides such as PI4P. Recent progress on the role of ORP-mediated lipid transport/countertransport at multiple MCSs in cellular functions will be also discussed.

Identification and analysis of non-specific lipid transfer protein family in tobacco

Plant non-specific lipid transfer proteins (nsLTPs) can transfer lipids in vitro , regulate plant growth and development, and respond to environmental abiotic and biotic stresses. In this study, 74 nsLTPs genes were identified from the genome of Nicotiana tabacum variety K326, and we analyzed multiple characteristics of these genes, including phylogeny, gene structures, conserved motifs, protein domains, chromosome locations, cis -elements in the promoter sequences, 3D structure, and the expression patterns under different hormones and abiotic stresses. The results revealed that nsLTPs in tobacco could be divided into eight types, including type I, II, III, IV, V, VII, VIII, and XIII, according to the interval and sequence similarity between the eight cysteines. The same types of NtLTPs had similar intron-exon patterns and conserved motifs, motif 2 and motif 3 were the characteristic motifs of NtLTPs family. In the process of evolution, fragment duplication dominated the expansion of the NtLTPs family. RNA-seq analysis after drought treatment revealed that the functional differentiation patterns of repeat gene pairs were diverse during evolution period. Promoter analysis showed that they contained a variety of cis-acting elements in response to light response, hormones, and abiotic stress. Furthermore, qRT-PCR demonstrated that NtLTPs family genes had different expression patterns in different tissues and organs of tobacco plants, which could respond to abiotic stresses such as drought, salt, and hormone treatments (IAA, GA, and SA etc.). These results provide a theoretical reference for the in-depth analysis of the functions of NtLTPs family genes and molecular breeding.

Lipid Binding Properties of Sec14‐like Homologues in the Yeast Saccharomyces cerevisiae

Lipids are hydrophobic molecules with several key functions in the cell. Due to the thermodynamic constraints placed upon lipids, they cannot freely diffuse across the cytosol. Their transport between two membrane compartments can be facilitated by lipid transfer proteins (LTPs). One of the most studied families of LTPs is S. cerevisiae’s Sec14p family. Proteins belonging to this group contain cellular retinaldehyde binding – triple functional (CRAL-TRIO) domain. In S. cerevisiae there are six members of this family, the founding member Sec14p and five of its homologues termed Sfh (Sec fourteen homologue) 1-5. The Sec14p has been extensively studied and it was determined that it possesses phosphatidylinositol (PI) and phosphatidylcholine (PC) binding and transfer activities (Bankaitis et al., 1990). Many of Sfh proteins have been implicated in various processes in cell, however, their exact roles remain to be discovered. Lipid binding and transfer by Sfh proteins were intensively studied (Li et al., 2000; Maeda et al., 2013; Tripathi et al., 2019; Holic et al., 2014; Pevalova et al., 2019) but some key questions remain open. Our objective is to determine lipids bound to S. cerevisiae’s Sec fourteen homologue proteins. For this we used HL-60 in situ lipid-binding assay, which employs co-incubation of permeabilized human HL-60 cells with recombinant protein of interest. Afterwards, the proteins are re-purified using affinity chromatography. The lipids extracted from re-isolated proteins are separated using thin layer chromatography and identified. Our results showed that Sfh1 binds PC and PI with approximately the same affinity. The remaining Sfh proteins, excluding Sfh5p, predominantly bind PI and neutral lipids (NLs). Sfh5 was found to bind only PI. Analysis of bound NLs identified squalene in Sfh2p as a likely binding partner and cholesterol in association with Sfh3p and Sfh4p. Moreover, it appears that both Sfh3p and Sfh4p can bind another NL in addition to cholesterol, which remains to be identified. Based on obtained results at least three out of five Sfh proteins are capable to bind NLs, predominantly sterols or precursors of the sterol biosynthetic pathway. It appears that binding of NLs is very specific, suggesting their crucial role in the function of Sfh proteins. Thus, the activity of some members of LTP family might be regulated by sterols or their precursors which in turn can connect and synchronize the sterol biosynthetic pathway with the phospholipid trafficking events. To identify the role of sterol binding by Sfh proteins in the physiology of the yeast cell, we will attempt to prepare mutants in sterol binding.

Non-specific Lipid Transfer Proteins in Legumes and Their Participation During Root-Nodule Symbiosis

Non-specific lipid transfer proteins (LTPs) constitute a large protein family in plants characterized by having a tunnel-like hydrophobic cavity, which allows them to transfer different lipid molecules. LTPs have been studied in various model plants including those of agronomic interest. Recent studies have demonstrated that LTPs play key functions in both biotic and abiotic stress. In plants-pathogen interaction, they act as either positive or negative regulators of defense responses. However, little is known about the roles of LTPs in symbiotic interactions, especially in root nodule symbiosis. Here, we performed a broad genome analysis of LTP family members in legumes and other important model plants, focusing on their possible roles in legume-rhizobium symbiosis. In silico analysis showed that legumes contain large LTP families, with at least 70 LTP members clustered into four clades. Although the structures of LTP genes and proteins are conserved among species, differences were observed between clades from different species. LTPs are widely expressed in different plant tissues. In general, genes of the LTP1 and LTP2 classes are highly expressed in shoot and reproductive tissues in all analyzed species. Furthermore, genes of the different classes are also expressed in roots inoculated with rhizobia and nodules of legumes. RT-qPCR expression profile analysis of seven PvLTP genes in common bean (Phaseolus vulgaris) revealed that these genes are differentially expressed during the early and late stages of nodulation and they are genetically regulated by PvRbohA. These findings provide insight into the putative roles of LTP family members in legume-rhizobium symbiosis and their possible interactions with RBOH-dependent ROS production.

Identification and functional analysis of the MdLTPG gene family in apple

Cuticular wax is synthesized from intracellular lipids that are exported by epidermal cells, and plant lipid transfer proteins (LTPs) play an important role in this process. The glycosylphosphatidylinositol (GPI)-anchored LTPs (LTPGs) are a large subgroup within the LTP family and function in lipid transport and wax formation. Although LTPG family members have been identified in several plant species, the LTPG gene family of apple (Malus domestica) remains uncharacterized. In this paper, we identified 26 potential LTPG genes by searching apple whole-genome annotation files using “GPI-anchored” and “lipid transferase” as keywords. Twenty of the 26 putative LTPG genes were confirmed as MdLTPG family members based on their subcellular localization predictions. The MdLTPGs were divided into four classes based on phylogenetic analysis and functional domain prediction. One member of each class was analyzed for subcellular localization, and all identified members were located on the plasma membrane. Most MdLTPG genes were induced by abiotic stress treatments such as low temperature, NaCl, and ABA. Finally, the MdLTPG17 protein was shown to interact with the lysine-rich arabinogalactan protein MdAGP18 to perform its function in wax transport during plant growth and development.

Structure of human ORP3 ORD reveals conservation of a key function and ligand specificity in OSBP-related proteins.

Human ORP3 belongs to the oxysterol-binding protein (OSBP) family of lipid transfer proteins and is involved in lipid trafficking and cell signaling. ORP3 localizes to the ER-PM interfaces and is implicated in lipid transport and focal adhesion dynamics. Here, we report the 2.6-2.7 Å structures of the ORD (OSBP-related domain) of human ORP3 in apo-form and in complex with phosphatidylinositol 4-phosphate. The ORP3 ORD displays a helix grip β-barrel fold with a deep hydrophobic pocket which is conserved in the OSBP gene family. ORP3 binds PI(4)P by the residues around tunnel entrance and in the hydrophobic pocket, whereas it lacks sterol binding due to the narrow hydrophobic tunnel. The heterologous expression of the ORDs of human ORP3 or OSBP1 rescued the lethality of seven ORP (yeast OSH1-OSH7) knockout in yeast. In contrast, the PI(4)P-binding site mutant of ORP3 did not complement the OSH knockout cells. The N-terminal PH domain and FFAT motif of ORP3 are involved in protein targeting but are not essential in yeast complementation. This observation suggests that the essential function conserved in the ORPs of yeast and human is mediated by PI(4)P-binding of the ORD domain. This study suggests that the non-vesicular PI(4)P transport is a conserved function of all ORPs in eukaryotes.