PtdIns) Transfer Proteins Research Articles

Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER-PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER-PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface-conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P2 hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER-PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P2 recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P2 or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER-PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER-PM junctions formed by Kv2 channel-VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

Novel Regulation of Lipid Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4‐Kinase

Membrane contact sites (MCSs) are regions in cells where membranes of two organelles are in close apposition. MCSs are proposed to facilitate non‐vesicular trafficking of lipids. Lipid transfer proteins (LTPs) are one of the most commonly‐found components in the MCS system and are speculated to mediate lipid transfer between membranes. However, both the biological functions of MCSs and the functional role of LTP in MCSs remain largely elusive. Herein, we examine in detail a previously‐proposed MCSs model, the ER‐Golgi/endosome MCS facilitating the phosphatidylserine (PtdSer) decarboxylation 2 (Psd2) pathway, which converts PtdSer into phosphatidylethanolamine (PtdEtn) in Saccharomyces cerevisiae. Particularly, we investigate the biological role of a yeast LTP, one of the yeast Phosphatidylinositol (PtdIns)‐Transfer Proteins (PITPs), Sfh4, and a PtdIns 4‐OH kinase Stt4, in this Psd2‐MCS system.We found that Psd2, Sfh4, and Stt4 are the only essential components facilitating Psd2‐dependent PtdEtn synthesis while other previously proposed elements constituting the Psd2‐MCS (Scs2, Scs22, and Pbi1) are dispensable. Surprisingly, neither the PtdIns‐transfer activity of Sfh4 nor its capacity to activate Stt4 is required to stimulate the Psd2 pathway. Instead, Sfh4 activates the Psd2 pathway via an Sfh4‐Psd2 physical interaction involving the F175 residue on the surface of Sfh4. Stt4 also displays physical interaction with Psd2 in an Sfh4‐independent manner. Instead of directly activating Psd2, Stt4 regulates the substrate pool accessible to Psd2, thereby indirectly controlling the Psd2‐mediated synthesis of PtdEtn. These results demonstrate that the ER‐endosomal MCS model is an inaccurate description of the Psd2 system in yeast, and provide an outstanding example where PITP biological function is uncoupled from its ‘canonical’ activity as a PtdIns transfer protein. Additionally, our findings reveal a novel mechanism in which a PITP and a PtdIns 4‐OH kinase regulate cellular phospholipid homeostasis.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (GM44530) and the Robert A. Welch Foundation (BE‐0117) to VAB. The authors declare no financial conflicts.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The interface between phosphatidylinositol transfer protein function and phosphoinositide signaling in higher eukaryotes

Phosphoinositides are key regulators of a large number of diverse cellular processes that include membrane trafficking, plasma membrane receptor signaling, cell proliferation, and transcription. How a small number of chemically distinct phosphoinositide signals are functionally amplified to exert specific control over such a diverse set of biological outcomes remains incompletely understood. To this end, a novel mechanism is now taking shape, and it involves phosphatidylinositol (PtdIns) transfer proteins (PITPs). The concept that PITPs exert instructive regulation of PtdIns 4-OH kinase activities and thereby channel phosphoinositide production to specific biological outcomes, identifies PITPs as central factors in the diversification of phosphoinositide signaling. There are two evolutionarily distinct families of PITPs: the Sec14-like and the StAR-related lipid transfer domain (START)-like families. Of these two families, the START-like PITPs are the least understood. Herein, we review recent insights into the biochemical, cellular, and physiological function of both PITP families with greater emphasis on the START-like PITPs, and we discuss the underlying mechanisms through which these proteins regulate phosphoinositide signaling and how these actions translate to human health and disease.

A Novel Multi‐domain Phosphatidylinositol Transfer Protein/Oxysterol Binding Protein Senses Specific Phosphoinositide Pools On Toxoplasma Dense Granules

The Apicomplexan Toxoplasma gondii is a common parasite of humans and other mammals and causes severe disease in immunocompromised individuals. Apicomplexa are obligate intracellular pathogens, and their infection of host cells requires the orchestrated secretion of proteins from several highly specialized secretory organelles, the regulation of which is incompletely understood. Studies in other eukaryotic systems have established that membrane trafficking through distal stages of the secretory pathway is regulated by phosphoinositide (PIP) signaling networks and that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are important components of these signaling circuits. We used PIP‐specific biosensors to map phosphoinositide pools on Toxoplasma membranes, and identified at least two distinct pools of PtdIns(4)P: one on the Golgi/early secretory membranes, and one associated with Apicomplexan‐specific secretory organelles called dense granules (GRA3(+)). Studies in yeast demonstrate that PITPs potentiate the activities of PtdIns kinases (PIKs) to produce privileged PIP pools on secretory membranes, and PtdIns(4)P‐mediated pro‐secretory activities are countered by the antagonistic activities of specific members of the oxysterol binding protein (OSBP) superfamily. We have identified a Toxoplasma multi‐domain protein, the PITP membrane protein (PIMP), that couples a START‐type PITP domain, a pleckstrin homology (PH) domain, and a C‐terminal OSBP domain. PIMP is a bona fide PITP that transfers PtdIns and phosphatidylcholine (PtdCho) between membranes in vitro, and potentiates PIK activity in vivo when expressed in yeast. Of the four known Toxoplasma PITP genes, it is the only one capable of doing so. The PIMP PH domain specifically binds PtdIns(4)P and PtdIns(4,5)P2 in vivo, and recognizes these pools on dense granules in intracellular parasites. Full length PIMP localizes to (GRA3+) dense granules, and this association is mediated by PH domain PIP binding. PIMP is a physical platform for integration of multiple lipid signals on specialized secretory membranes in Toxoplasma and is the first known example of a PITP coupled to an OSBP domain. As dense granules are unique to Apicomplexa and required to support intracellular proliferation of the parasites, dense granule signaling networks would make good therapeutic targets for Apicomplexan infections.Support or Funding InformationThis research was funded by NIH grant R01‐GM112591, RO1‐GM44530 and grant BE‐0017 from the Robert A. Welch Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Golgi Lipid Signaling Pathway Controls Apical Golgi Distribution and Cell Polarity during Neurogenesis

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) stimulate PtdIns-4-P synthesis and signaling in eukaryotic cells, but to what biological outcomes such signaling circuits are coupled remains unclear. Herein, we show that two highly related StART-like PITPs, PITPNA and PITPNB, act in a redundant fashion to support development of the embryonic mammalian neocortex. PITPNA/PITPNB do so by driving PtdIns-4-P-dependent recruitment of GOLPH3, and likely ceramide transferprotein (CERT), to Golgi membranes with GOLPH3 recruitment serving to promote MYO18A- and F-actin-directed loading of the Golgi network to apical processes of neural stem cells (NSCs). We propose the primary role for PITP/PtdIns-4-P/GOLPH3/CERT signaling in NSC Golgi is not in regulating bulk membrane trafficking but in optimizing apically directed membrane trafficking and/or apical membrane signaling during neurogenesis.

The Use of Fluorescently Labeled Lipids to Determine Dynamics of Protein Mediated In Vitro Lipid Transfer

In recent years, phosphatidylinositol (PtdIns) transfer proteins (PITPs) have been established as essential regulators of phosphoinositide signaling outcomes in eukaryotic cells. The key function of these proteins is to mediate a PtdIns/phosphatidylcholine (PtdCho) exchange reaction. This function is central to their abilities to regulate lipid signaling. Despite its importance the mechanistic details of this lipid exchange remain largely undetermined. In order to decipher some aspects of the exchange reaction, we have modified an in vitro lipid transfer method first described by Somerharju et al. (Biochemistry, 1984). By utilizing pyrene-labeled PtdCho and PtdIns lipid species the membrane extraction and lipid transfer mediated by the mammalian StART-like PITP, PITPα (and other lipid transfer proteins) has been fluorescently monitored. We have also further modified the method to include the measuring of protein mediated transfer of the intrinsically fluorescent sterol, dehydroergosterol (DHE). Using the method, we have studied how point mutations in the binding pocket of PITPα and the yeast Sec14-like PtdIns transfer protein, Sfh3, affect PC/PI and DHE binding and transfer respectively. The advantages of using fluorescently labeled lipids to measure protein mediated transfer includes time-resolved data acquisition and greater sensitivity compared to other established methods. The possible artefacts introduced by the pyrene label can, however, not be dismissed. To address this, we have used a well-established in vitro radiolabeled transfer assay to verify and validate the results obtained using the fluorescence assay. These results will help to increase the understanding of how cells utilize PITPs in regulating lipid signaling cascades.

Sec14-like phosphatidylinositol transfer proteins and the biological landscape of phosphoinositide signaling in plants

Phosphoinositides and soluble inositol phosphates are essential components of a complex intracellular chemical code that regulates major aspects of lipid signaling in eukaryotes. These involvements span a broad array of biological outcomes and activities, and cells are faced with the problem of how to compartmentalize and organize these various signaling events into a coherent scheme. It is in the arena of how phosphoinositide signaling circuits are integrated and, and how phosphoinositide pools are functionally defined and channeled to privileged effectors, that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are emerging as critical players. As plant systems offer some unique advantages and opportunities for study of these proteins, we discuss herein our perspectives regarding the progress made in plant systems regarding PITP function. We also suggest interesting prospects that plant systems hold for interrogating how PITPs work, particularly in multi-domain contexts, to diversify the biological outcomes for phosphoinositide signaling. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein.

Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and-B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER-PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER-PM contact zones with a possible link to a devastating human disease.

Sec14-like phosphatidylinositol-transfer proteins and diversification of phosphoinositide signalling outcomes.

The physiological functions of phosphatidylinositol (PtdIns)-transfer proteins (PITPs)/phosphatidylcholine (PtdCho)-transfer proteins are poorly characterized, even though these proteins are conserved throughout the eukaryotic kingdom. Much of the progress in elucidating PITP functions has come from exploitation of genetically tractable model organisms, but the mechanisms for how PITPs execute their biological activities remain unclear. Structural and molecular dynamics approaches are filling in the details for how these proteins actually work as molecules. In the present paper, we discuss our recent work with Sec14-like PITPs and describe how PITPs integrate diverse territories of the lipid metabolome with phosphoinositide signalling.

Dimeric Sfh3 has structural changes in its binding pocket that are associated with a dimer–monomer state transformation induced by substrate binding

Phosphorylated derivatives of phosphatidylinositol (PtdIns), also called phosphoinositides (PIPs), are basic components of membrane-associated signalling systems. A family of PtdIns-transfer proteins (PITPs) called the Sec14 family have been predicted to form a set of functional modules that can sense different types of lipid metabolism and transmit the information to the PIP signalling system. In eukaryotic cells, the Sec14 family exhibits a wide diversity of activity, but the structural basis of this diversity remains unclear. In the present study, the dimeric structure of Sfh3 (Sec14 family homologue 3 in yeast) is reported for the first time and differs from the Sec14 proteins reported to date, all of which are monomeric. Some variations in the binding pocket of Sfh3 were observed and the dimer interface was identified and proposed to provide a link between dimer-monomer state changes and PtdIns binding. Together, these structural changes and the oligomeric state transformation of Sfh3 support ideas of diversity within the Sec14 family and provide some new clues to function.

Thoughts on Sec14-like nanoreactors and phosphoinositide signaling

Thoughts on Sec14-like nanoreactors and phosphoinositide signaling

A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p nullizygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.