The Sec14-superfamily and the regulatory interface between phospholipid metabolism and membrane trafficking

A central principle of signal transduction is the appropriate control of the process so that relevant signals can be detected with fine spatial and temporal resolution. In the case of lipid-mediated signaling, organization and metabolism of specific lipid mediators is an important aspect of such control. Herein, we review the emerging evidence regarding the roles of Sec14-like phosphatidylinositol transfer proteins (PITPs) in the action of intracellular signaling networks; particularly as these relate to membrane trafficking. Finally, we explore developing ideas regarding how Sec14-like PITPs execute biological function. As Sec14-like proteins define a protein superfamily with diverse lipid (or lipophile) binding capabilities, it is likely these under-investigated proteins will be ultimately demonstrated as a ubiquitously important set of biological regulators whose functions influence a large territory in the signaling landscape of eukaryotic cells.     

Phosphatidylinositol transfer proteins and cellular nanoreactors for lipid signaling

Membrane lipids function as structural molecules, reservoirs for second messengers, membrane platforms that scaffold protein assembly and regulators of enzymes and ion channels. Such diverse lipid functions contribute substantially to cellular mechanisms for fine-tuning membrane-signaling events. Meaningful coordination of these events requires exquisite spatial and temporal control of lipid metabolism and organization, and reliable mechanisms for specifically coupling these parameters to dedicated physiological processes. Recent studies suggest such integration is linked to the action of phosphatidylinositol transfer proteins that operate at the interface of the metabolism, trafficking and organization of specific lipids.     

Phosphatidylinositol transfer proteins and functional specification of lipid signaling pools

The diversity of lipid species in biological membranes testifies to the multiple roles of these molecules as structural units, precursors to second messengers, as scaffolding units that impose spatial and temporal regulation on assembly of proteins, and as regulators of the catalytic activities of proteins. Such diverse lipid functions must be appropriately coordinated so that these can be specifically and appropriately coupled to dedicated biological processes. Evidence from multiple sources is building towards a concept where Sec14-like PITPs are specific components of lipid metabolic nanoreactors and, in this capacity, help impose a functional specification of lipid signaling pools.     

The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes

Phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs) remain largely functionally uncharacterized, despite the fact that they are highly conserved and are found in all eukaryotic cells thus far examined by biochemical or sequence analysis approaches. The available data indicate a role for PITPs in regulating specific interfaces between lipid-signaling and cellular function. In this regard, a role for PITPs in controlling specific membrane trafficking events is emerging as a common functional theme. However, the mechanisms by which PITPs regulate lipid-signaling and membrane-trafficking functions remain unresolved. Specific PITP dysfunctions are now linked to neurodegenerative and intestinal malabsorption diseases in mammals, to stress response and developmental regulation in higher plants, and to previously uncharacterized pathways for regulating membrane trafficking in yeast and higher eukaryotes, making it clear that PITPs are integral parts of a highly conserved signal transduction strategy in eukaryotes. Herein, we review recent progress in deciphering the biological functions of PITPs, and discuss some of the open questions that remain.     

Sec14 related proteins in yeast

Lipid transport between membranes of eukaryotic organisms represents an essential aspect of organelle biogenesis. This transport must be strictly selective and directional to assure specific lipid composition of individual membranes. Despite the intensive research effort in the last few years, our understanding of how lipids are sorted and moved within cells is still rather limited. Evidence indicates that at least some of the mechanisms generating and maintaining non-random distribution of lipids in cells are linked to the action of phosphatidylinositol transfer proteins (PITPs). The major PITP in yeast Saccharomyces cerevisiae, Sec14p, is essential in promoting Golgi secretory function by modulating of its membrane lipid composition. This review focuses on a group of five yeast proteins that share significant sequence homology with Sec14p. Based on this sequence identity, they were termed Sfh (Sec fourteen homologue) proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism, phosphoinositide mediated signaling and membrane trafficking.     

Biophysics of ceramide signaling: interaction with proteins and phase transition of membranes

Ceramides have been implied in intracellular signal transduction systems regulating cellular differentiation, activation, survival and apoptosis and thus appear capable of changing the life style of virtually any cell type. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of diverse cellular responses to exogenous stimuli. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus and cell-type specific and depends on the subcellular topology of its production. However, little is understood about the molecular mode of ceramide action. In particular, in lieu of a defined ceramide binding motif it is not clear how ceramide would directly interact with putative target signaling proteins. This article proposes two modes of ceramide action. First, a protruding alkyl chain of ceramide may interact with a hydrophobic cavity of a signaling protein providing a lipid anchor to attach proteins to membranes. Second, the generation of ceramide generally increases the volume of hydrocarbon chains within the lipid bilayer thereby enhancing its propensity of to form a hexagonal II phase (Hex II). Besides the generation of a hydrophobic interaction site for proteins local hexagonal phase II formation can also change the membrane fluidity and permeability, which may impinge on membrane fusion processes, solubilization of detergent-resistant signaling rafts, or membrane receptor internalization. Thus, ceramide production by sphingomyelinases (SMase) can play a pivotal signaling role through direct interaction with signaling proteins or through facilitating the formation and trafficking of signal transduction complexes.     

Self-assemblies of Rab- and Arf-family small GTPases on lipid bilayers in membrane tethering

Small GTPases of the Ras superfamily, which include Ras-, Rho-, Rab-, Arf-, and Ran-family isoforms, are generally known to function as a nucleotide-dependent molecular switch in eukaryotic cells. In the GTP-loaded forms, they selectively recruit their cognate interacting proteins or protein complexes, termed “effectors,” to the cytoplasmic face of subcellular membrane compartments, thereby switching on the downstream effector functions, which are vital for fundamental cellular events, such as cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. Nevertheless, in addition to acting as the classic nucleotide-dependent switches for the effectors, recent studies have uncovered that small GTPases themselves can be self-assembled specifically into homo-dimers or higher-order oligomers on membranes, and these assembly processes are likely responsible for their physiological functions. This Review focuses particularly on the self-assembly processes of Rab- and Arf-family isoforms during membrane tethering, the most critical step to ensure the fidelity of membrane trafficking. A summary of the current experimental evidence for self-assemblies of Rab and Arf small GTPases on lipid bilayers in chemically defined reconstitution system is provided     

Dynamics of lipid transfer by phosphatidylinositol transfer proteins in cells

Of many lipid transfer proteins identified, all have been implicated in essential cellular processes, but the activity of none has been demonstrated in intact cells. Among these, phosphatidylinositol transfer proteins (PITP) are of particular interest as they can bind to and transfer phosphatidylinositol (PtdIns)--the precursor of important signalling molecules, phosphoinositides--and because they have essential functions in neuronal development (PITPalpha) and cytokinesis (PITPbeta). Structural analysis indicates that, in the cytosol, PITPs are in a 'closed' conformation completely shielding the lipid within them. But during lipid exchange at the membrane, they must transiently 'open'. To study PITP dynamics in intact cells, we chemically targeted their C95 residue that, although non-essential for lipid transfer, is buried within the phospholipid-binding cavity, and so, its chemical modification prevents PtdIns binding because of steric hindrance. This treatment resulted in entrapment of open conformation PITPs at the membrane and inactivation of the cytosolic pool of PITPs within few minutes. PITP isoforms were differentially inactivated with the dynamics of PITPbeta faster than PITPalpha. We identify two tryptophan residues essential for membrane docking of PITPs.     

Control of vesicular trafficking by Rho GTPases

Although vesicular trafficking is essential for a large variety of cellular processes, the regulation of vesicular trafficking is still poorly understood. Members of the Rho family of small GTPases have recently emerged as important control elements of many stages of vesicular trafficking, providing new insight into the regulation of these events. We will discuss the diverse roles played by Rho proteins in membrane trafficking and focus on the biological implications of these functions.     

Biochemical and biological functions of class I phosphatidylinositol transfer proteins

Phosphoinositides function in a diverse array of cellular activities. They include a role as substrate for lipid kinases and phospholipases to generate second messengers, regulators of the cytoskeleton, of enzymes and of ion channels, and docking sites for reversible recruitment of proteins to membranes. Mammalian phosphatidylinositol transfer proteins, PITPalpha and PITPbeta are paralogs that share 77% sequence identity and contain a hydrophobic cavity that can sequester either phosphatidylinositol or phosphatidylcholine. A string of 11 amino acid residues at the C-terminal acts as a "lid" which shields the lipid from the aqueous environment. PITPs in vitro can facilitate inter-membrane lipid transfer and this requires the movement of the "lid" to allow the lipid cargo to be released. Thus PITPs are structurally designed for delivering lipid cargo and could thus participate in cellular events that are dependent on phosphatidylinositol or derivatives of phosphatidylinositol. Phosphatidylinositol, the precursor for all phosphoinositides is synthesised at the endoplasmic reticulum and its distribution to other organelles could be facilitated by PITPs. Here we highlight recent studies that report on the three-dimensional structures of the different PITP forms and suggest how PITPs are likely to dock at the membrane surface for lipid delivery and extraction. Additionally we discuss whether PITPs are important regulators of sphingomyelin metabolism, and finally describe recent studies that link the association of PITPs with diverse functions including membrane traffic at the Golgi, neurite outgrowth, cytokinesis and stem cell growth.     

Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium

Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium.     

泛素化介导的非蛋白质降解功能

泛素因标记被26 S蛋白酶体降解的蛋白质而著名．然而近几年发现,泛素作用远不止此,不仅具有参与蛋白质降解这一重要“传统作用”,还起着比先前想象更多变的、更精美的细胞调控作用,是非常重要的细胞过程的多层面调节因子,具有许多重要的非蛋白质降解功能,包括DNA损伤修复、DNA复制、信号传导、转录调节、膜运输、胞吞、蛋白激酶活化、染色质重塑和病毒芽殖．这些功能涉及多聚泛素化和单泛素化及多泛素化．因此,泛素化异常可能涉及疾病的发生和发展．对这些功能的了解可以拓展人们对泛素的认识,有助于对多种细胞过程的深入理解,也有助于相关新药的研发．     

Probing lipid-protein interactions using lipid microarrays

Lipids are central to the regulation and control of several cellular functions. They form many of the important structural features of cells, and are critical members of cellular signal transduction pathways. Cellular dysfunction is often caused by errors in lipid signaling; therefore, the proteins that interact with, synthesize or metabolize the lipids are potential therapeutic targets. Characterizing the contingent of cellular lipids and their abundance and how this is associated with disease will facilitate understanding how to intervene to correct diseases caused by dysfunctional lipid signaling. Since lipid-signaling networks involve several classes of proteins it is essential to determine the identity and role of these proteins in order to understand the networks. These proteins may be receptors, effectors, transporters or enzymes. We present tools, specifically, a lipid microarray platform, to uncover lipid-binding effector proteins that function in lipid signaling pathways. Lipid microarrays will allow researchers to obtain a comparable fingerprint of the proteins from a cell or tissue that bind to lipids, and also enable the identification of functionally important lipid-binding proteins. By applying a systematic approach to the quantification of lipid-protein interactions, lipid microarrays will provide an integrated knowledge base for the human lipidome. These tools have the potential to identify and validate targets to improve personalized medicine and health.     

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.     

Sac1, a lipid phosphatase at the interface of vesicular and nonvesicular transport

The lipid phosphatase Sac1 dephosphorylates phosphatidylinositol 4‐phosphate (PI4P), thereby holding levels of this crucial membrane signaling molecule in check. Sac1 regulates multiple cellular processes, including cytoskeletal organization, membrane trafficking and cell signaling. Here, we review the structure and regulation of Sac1, its roles in cell signaling and development and its links to health and disease. Remarkably, many of the diverse roles attributed to Sac1 can be explained by the recent discovery of its requirement at membrane contact sites, where its consumption of PI4P is proposed to drive interorganelle transfer of other cellular lipids, thereby promoting normal lipid homeostasis within cells.      

Courier service for phosphatidylinositol: PITPs deliver on demand

Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPβ and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.     

PI transfer protein: the specific recognition of phospholipids and its functions

Phosphatidylinositol transfer proteins (PITPs) can bind specifically and transfer a single phosphatidylinositol (PI) molecule between phospholipid membranes in an ATP-independent manner in vitro. PITPs exist in all the eukaryotic systems from yeast to human. PITP plays an essential role in intracellular vesicle flow and inositol lipid signaling. The crystal structure of yeast PITP Sec14p reveals a large hydrophobic pocket to accommodate the acyl chains of phospholipid molecules. At the opening of the pocket, a hydrogen bond network may render Sec14p the binding specificity to PI molecules. The structure suggests that the PI-binding ability may play an important role in the in vivo function of PITPs.     

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.     

Trafficking of phosphatidylinositol by phosphatidylinositol transfer proteins

PtdIns is synthesized at the endoplasmic reticulum and its intracellular distribution to other organelles can be facilitated by lipid transfer proteins [PITPs (phosphatidylinositol transfer proteins)]. In this review, I summarize the current understanding of how PITPs are regulated by phosphorylation, how can they dock to membranes to exchange their lipid cargo and how cells use PITPs in signal transduction and membrane delivery. Mammalian PITPs, PITPalpha and PITPbeta, are paralogous genes that are 94% similar in sequence. Their structural design demonstrates that they can sequester PtdIns or PtdCho (phosphatidylcholine) in their hydrophobic cavity. To deliver the lipid cargo to a membrane, PITP has to undergo a conformational change at the membrane interface. PITPs have a higher affinity for PtdIns than PtdCho, which is explained by hydrogen-bond contacts between the inositol ring of PtdIns and the side-chains of four amino acid residues, Thr59, Lys61, Glu86 and Asn90, in PITPs. Regardless of species, these residues are conserved in all known PITPs. PITP transfer activity is regulated by a conserved serine residue (Ser166) that is phosphorylated by protein kinase C. Ser166 is only accessible for phosphorylation when a conformational change occurs in PITPs while docking at the membrane interface during lipid transfer, thereby coupling regulation of activity with lipid transfer function. Biological roles of PITPs include their ability to couple phospholipase C signalling to neurite outgrowth, cell division and stem cell growth.     

Cinderella story: PI4P goes from precursor to key signaling molecule

Abstract

Phosphatidylinositol lipids are signaling molecules involved in nearly all aspects of cellular regulation. Production of phosphatidylinositol 4-phosphate (PI4P) has long been recognized as one of the first steps in generating poly-phosphatidylinositol phosphates involved in actin organization, cell migration, and signal transduction. In addition, progress over the last decade has brought to light independent roles for PI4P in membrane trafficking and lipid homeostasis. Here, we describe recent advances that reveal the breadth of processes regulated by PI4P, the spectrum of PI4P effectors, and the mechanisms of spatiotemporal control that coordinate crosstalk between PI4P and cellular signaling pathways.