Yeast Phosphatidylinositol Transfer Protein Research Articles

Synthesis of a photocleavable bola-phosphatidylcholine

Phosphatidylinositol transfer proteins (PITPs) are ubiquitous in eukaryotes and are involved in the regulation of phospholipid metabolism, membrane trafficking, and signal transduction. Sec14 is a yeast PITP that has been shown to transfer phosphatidylinositol (PI) or phosphatidylcholine (PC) from the endoplasmic reticulum to the Golgi. It is now believed that Sec14 may play a greater role than just shuttling PI and PC throughout the cell. Genetic evidence suggests that retrieval of membrane-bound PI by Sec14 also manages to present PI to the phosphatidylinositol-4-kinase, Pik1, to generate phosphatidylinositol-4-phosphate, PI(4)P. To test this hypothetical model, we designed a photocleavable bolalipid to span the entire membrane, having one phosphatidylcholine or phosphatidylinositol headgroup on each leaflet connected by a photocleavable diacid. Sec14 should not be able to present the bola-PI to Pik1 for phosphorylation as the head group will be difficult to lift from the bilayer as it is tethered on the opposite leaflet. After photocleavage the two halves would behave as a normal phospholipid, thus phosphorylation by Pik1 would resume. We report here the synthesis of a photocleavable bola-PC, a precursor to the desired bola-PI. The mono-photocleavable bola-PC lipid was designed to contain two glycerol molecules with choline head groups connected through a phosphodiester bond at the sn3 position. Each glycerol was acylated with palmitic acid at the sn1 position. These two glycerol moieties were then connected through their respective sn2 hydroxyls via a photocleavable dicarboxylic acid containing a nitrophenyl ethyl photolabile protecting group. The bola-PC and its precursors were found to undergo efficient photocleavage when irradiated in solution or in vesicles with 365 nm light for two minutes. Treatment of the bola-PC with a mutant phospholipase D and myo-inositol produced a mono-inositol bola-PC-PI.

Sec14-like-Lipid-Binding-4 Enables Hemoglobin Clearance By Liver Sinusoidal Endothelial Cells

Hemolysis of banked red blood cells (RBC storage lesion) contributes to lack of therapeutic benefit and higher mortality following transfusion therapy in patients. However, the molecular mechanism underlying storage lesion remains poorly understood. Using a multiple-ancestry genome-wide association analysis among blood donors, our group has recently identified Sec14-like-lipid-binding-4 (Sec14L4) protein to be associated with increased rate of hemolysis following blood storage. Although Sec14L4 is highly similar to yeast phosphatidylinositol transfer protein Sec14, which is known to be involved in the synthesis of golgi-derived transport vesicles, the exact function of Sec14L4 and how it promotes RBC storage lesions remains unknown. Our initial studies using fluorescence microscopy in our newly generated Sec14L4 null mice revealed significant enrichment of Sec14L4 in the live sinusoidal endothelial cells (LSECs), which positively correlated with elevated levels of iron in the liver. Scanning and transmission electron micrographs revealed impaired actin cytoskeleton organization in LSECs of Sec14L4 null mice, which was associated with upregulation of major GTPases RhoA, RhoB and Rac1 activity. The impairment of actin cytoskeletal organization in Sec14L4 null mice lead to impaired vesicle trafficking in LSECs and higher retention of RBCs in the liver sinusoids compared to control mice. These data are the first to demonstrate a previously unrecognized role for Sec14L4 protein in LSEC mediated endocytic trafficking by promoting actin polymerization. These findings also suggest that Sec14L4 may regulate RBC storage lesion through impaired hemoglobin clearance by LSECs secondary to cytoskeletal impairment.

Structural basis of ligand recognition and transport by Sfh2, a yeast phosphatidylinositol transfer protein of the Sec14 superfamily.

Sec14-like phosphatidylinositol transfer proteins (PITPs) are involved in lipid metabolism and phosphatidylinositol 4-phosphate signaling by transporting phosphatidylinositol (PI) and a secondary ligand between the organellar membranes in eukaryotes. Yeast Sfh2 is a PITP that transfers PI and squalene without phosphatidylcholine transfer activity. To investigate the structural determinants for ligand specificity and transport in Sfh2, crystal structures of Sfh2 in complex with PI and squalene were determined at 1.5 and 2.4 Å resolution, respectively. The inositol head group of PI is recognized by highly conserved residues around the pocket entrance. The acyl chains of PI bind into a large hydrophobic cavity. Squalene is accommodated in the bottom of the cavity entirely by hydrophobic interactions. The binding of PI and squalene are mutually exclusive due to their overlapping binding sites, correlating with the role in lipid exchange. The binding mode of PI is well conserved in Sfh family proteins. However, squalene binding is unique to the Sfh2 homolog due to the specific hydrophobic residues forming a shape-complementary binding pocket. Recombinant apo Sfh2 forms a homodimer in vitro by the hydrophobic interaction of the gating α10-α11 helices in an open conformation. Ligand binding closes the lid and dissociates the dimer into monomers. This study reveals the structural determinants for the recognition of the conserved PI and a secondary ligand, squalene, and provides implications for the lipid-transfer function of Sfh2.

Yeast phosphatidylinositol transfer protein Pdr17 does not require high affinity phosphatidylinositol binding for its cellular function

Yeast phosphatidylinositol transfer protein (PITP) Pdr17 is an essential component of the complex required for decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine (PE) at a non-mitochondrial location. According to current understanding, this process involves the transfer of PS from the endoplasmic reticulum to the Golgi/endosomes. We generated a Pdr17E237A, K269A mutant protein to better understand the mechanism by which Pdr17p participates in the processes connected to the decarboxylation of PS to PE. We show that the Pdr17E237A, K269A mutant protein is not capable of binding phosphatidylinositol (PI) using permeabilized human cells, but still retains the ability to transfer PI between two membrane compartments in vitro. We provide data together with molecular models showing that the mutations E237A and K269A changed only the lipid binding cavity of Pdr17p and not its surface properties. In contrast to Pdr16p, a close homologue, the ability of Pdr17p to bind PI is not required for its major cellular function in the inter-membrane transfer of PS. We hypothesize that these two closely related yeast PITPs, Pdr16p and Pdr17p, have evolved from a common ancestor. Pdr16p fulfills those role(s) in which the ability to bind and transfer PI is required, while Pdr17p appears to have adapted to a different role which does not require the high affinity binding of PI, although the protein retains the capacity to transfer PI. Our results indicate that PITPs function in complex ways in vivo and underscore the need to consider multiple PITP parameters when studying these proteins in vitro.

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.

PITPs as targets for selectively interfering with phosphoinositide signaling in cells.

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Trans-Golgi Network and Endosome Dynamics Connect Ceramide Homeostasis with Regulation of the Unfolded Protein Response and TOR Signaling in Yeast

Synthetic genetic array analyses identify powerful genetic interactions between a thermosensitive allele (sec14-1(ts)) of the structural gene for the major yeast phosphatidylinositol transfer protein (SEC14) and a structural gene deletion allele (tlg2Delta) for the Tlg2 target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor. The data further demonstrate Sec14 is required for proper trans-Golgi network (TGN)/endosomal dynamics in yeast. Paradoxically, combinatorial depletion of Sec14 and Tlg2 activities elicits trafficking defects from the endoplasmic reticulum, and these defects are accompanied by compromise of the unfolded protein response (UPR). UPR failure occurs downstream of Hac1 mRNA splicing, and it is further accompanied by defects in TOR signaling. The data link TGN/endosomal dynamics with ceramide homeostasis, UPR activity, and TOR signaling in yeast, and they identify the Sit4 protein phosphatase as a primary conduit through which ceramides link to the UPR. We suggest combinatorial Sec14/Tlg2 dysfunction evokes inappropriate turnover of complex sphingolipids in endosomes. One result of this turnover is potentiation of ceramide-activated phosphatase-mediated down-regulation of the UPR. These results provide new insight into Sec14 function, and they emphasize the TGN/endosomal system as a central hub for homeostatic regulation in eukaryotes.

Conformational dynamics of the major yeast phosphatidylinositol transfer protein sec14p: insight into the mechanisms of phospholipid exchange and diseases of sec14p-like protein deficiencies.

Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A(10)/T(4)/A(11) helical gate, and that "state-of-closure" of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F(212), Y(213), K(239), I(240), and I(242)) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B(1)LB(2) and A(12)LT(5) substructures) through which conformational information is transduced to the hinge. The (114)TDKDGR(119) motif of B(1)LB(2) plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.

Conformational Dynamics of the Major Yeast Phosphatidylinositol Transfer Protein Sec14p: Insight into the Mechanisms of Phospholipid Exchange and Diseases of Sec14p-Like Protein Deficiencies

Conformational Dynamics of the Major Yeast Phosphatidylinositol Transfer Protein Sec14p: Insight into the Mechanisms of Phospholipid Exchange and Diseases of Sec14p-Like Protein Deficiencies

Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi

The budding yeast Saccharomyces cerevisiae contains a family of Arf (ADP-ribosylation factor) GTPase activating protein (GAP) proteins with the Gcs1 + Age2 ArfGAP pair providing essential overlapping function for the movement of transport vesicles from the trans-Golgi network. We have generated a temperature-sensitive but stable version of the Gcs1 protein that is impaired only for trans-Golgi transport and find that deleterious effects of this enfeebled Gcs1-4 mutant protein are relieved by increased gene dosage of the gcs1-4 mutant gene itself or by the SFH2 gene (also called CSR1), encoding a phosphatidylinositol transfer protein (PITP). This effect was not seen for the SEC14 gene, encoding the founding member of the yeast PITP protein family, even though the Gcs1 and Age2 ArfGAPs are known to be downstream effectors of Sec14-mediated activity for trans-Golgi transport. Sfh2-mediated suppression of inadequate Gcs1-4 function depended on phospholipase D, whereas inadequate Gcs1-4 activity was relieved by increasing levels of diacylglycerol (DAG). Recombinant Gcs1 protein was found to bind certain phospholipids but not DAG. Our findings favor a model of Gcs1 localization through binding to specific phospholipids and activation of ArfGAP activity by DAG-mediated membrane curvature as the transport vesicle is formed. Thus, ArfGAPs are subject to both temporal and spatial regulation that is facilitated by Sfh2-mediated modulation of the lipid environment.

Regulation of phospholipid biosynthesis by phosphatidylinositol transfer protein Sec14p and its homologues

Transcription of yeast phospholipid biosynthesis structural genes, which contain an inositol-sensitive upstream activating sequence in their promoters, responds to the availability of the soluble precursors inositol and choline and to changes in phospholipid metabolism. The INO1 gene is deregulated (derepressed when inositol is present) under the conditions of increased phosphatidylcholine (PtdCho) turnover, as occurs in the sec14Delta cki1Delta strain (SEC14 encodes the major yeast phosphatidylinositol transfer protein; CKI1 encodes choline kinase of the cytidine diphosphate choline pathway of PtdCho biosynthesis). Five proteins (Sfhp) share sequence homology with phosphatidylinositol transfer protein Sec14p. Two (Sfh2p and Sfh4p), when overexpressed largely complement the otherwise essential Sec14p requirement concerning growth and secretion. In this study, we analysed the ability of Sec14 homologues to correct the defect in regulation of phospholipid biosynthesis resulting from defective or missing Sec14p. We also analysed how PtdCho turnover relates to the transcriptional regulation of phospholipid biosynthesis. The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal of phospholipase D activity corrected the aberrant INO1 gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of the INO1 gene. Overall, the results support the model in which high phosphatidic acid levels lead to derepression of the genes of phospholipid biosynthesis [Henry, S.A. & Patton-Vogt, J.L. (1998) Prog. Nucleic Acid Res. Mol. Biol.61, 133-179].

Ligand specificity in the CRAL-TRIO protein family.

Intracellular trafficking of hydrophobic ligands is often mediated by specific binding proteins. The CRAL-TRIO motif is common to several lipid binding proteins including the cellular retinaldehyde binding protein (CRALBP), the alpha-tocopherol transfer protein (alpha-TTP), yeast phosphatidylinositol transfer protein (Sec14p), and supernatant protein factor (SPF). To examine the ligand specificity of these proteins, we measured their affinity toward a variety of hydrophobic ligands using a competitive [(3)H]-RRR-alpha-tocopherol binding assay. Alpha-TTP preferentially bound RRR-alpha-tocopherol over all other tocols assayed, exhibiting a K(d) of 25 nM. Binding affinities of other tocols for alphaTTP closely paralleled their ability to inhibit in vitro intermembrane transfer and their potency in biological assays. All other homologous proteins studied bound alpha-tocopherol but with pronouncedly weaker (> 10-fold) affinities than alpha-TTP. Sec14p demonstrated a K(d) of 373 nM for alpha-tocopherol, similar to that for its native ligand, phosphatidylinositol (381 nM). Human SPF had the highest affinity for phosphatidylinositol (216 nM) and gamma-tocopherol (268 nM) and significantly weaker affinity for alpha-tocopherol (K(d) 615 nM). SPF bound [(3)H]-squalene more weakly (879 nM) than the other ligands. Our data suggest that of all known CRAL-TRIO proteins, only alphaTTP is likely to serve as the physiological mediator of alpha-tocopherol's biological activity. Further, ligand promiscuity observed within this family suggests that caution should be exercised when suggesting protein function(s) from measurements utilizing a single ligand.

Crystal Structure of the Human Supernatant Protein Factor

Supernatant protein factor (SPF) promotes the epoxidation of squalene catalyzed by microsomes. Several studies suggest its in vivo role in the cholesterol biosynthetic pathway by a yet unknown mechanism. SPF belongs to a family of lipid binding proteins called CRAL_TRIO, which include yeast phosphatidylinositol transfer protein Sec14 and tocopherol transfer protein TTP. The crystal structure of human SPF at a resolution of 1.9 Å reveals a two domain topology. The N-terminal 275 residues form a Sec14-like domain, while the C-terminal 115 residues consist of an eight-stranded jelly-roll barrel similar to that found in many viral protein structures. The ligand binding cavity has a peculiar horseshoe-like shape. Contrary to the Sec14 crystal structure, the lipid-exchange loop is in a closed conformation, suggesting a mechanism for lipid exchange.

Activity of Specific Lipid-regulated ADP Ribosylation Factor-GTPase–activating Proteins Is Required for Sec14p-dependent Golgi Secretory Function in Yeast

Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these “bypass Sec14p” mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive. We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase–activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function. In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function. These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle.

Activity of specific lipid-regulated ADP ribosylation factor-GTPase-activating proteins is required for Sec14p-dependent Golgi secretory function in yeast.

Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these "bypass Sec14p" mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive. We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase-activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function. In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function. These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle.

Plant SEC14-like proteins controlling a phosphoinositide traffic jam

Phosphatidylinositol transfer proteins (PITPs) are defined by their ability to transfer phosphatidylinositol or a phosphatidylcholine monomer between membrane bilayers in vitro. Studies in mammalian systems have suggested that PITPs play a key role in controlling the activities of various inositol lipid-signalling pathways by regulating the production of some phosphoinositides. These inositol-containing phospholipids belong to a large and heterogeneous group of lipids with structural and key regulatory roles in a wide range of cellular signalling events. Recently, several plant proteins similar to yeast PITP (SEC14) have also been identified and characterized.

Endogenous Antisense

A search for genes in Lotus japonicus related to a nodule-specific transcript LjNOD16 led to the identification of a gene family encoding proteins with an NH 2 -domain homologous to the yeast phosphatidylinositol transfer protein (PITP). The predicted proteins also have the conserved COOH-domain with two α helices and a positively charged COOH-terminus found in Nlj16 (the protein encoded by LjNOD16 ), which is involved in membrane targeting. The gene from which Nlj16 is derived ( LjPLP-IV ) had an unusual expression pattern, detected by Northern analysis, with sense transcripts expressed in flowers and antisense transcripts expressed in nodules. Analysis of the gene showed a bidirectional promoter in intron 10 that was able to direct expression of a reporter gene oriented in either the sense or antisense direction in nodule tissue. Analysis of the ability of the full-length sense-directed transcript of LjPLP-IV to rescue yeast deficient in PITP suggested that the COOH-terminal Nlj16 domain inhibited PITP activity of LjPLP-IV. The PITP activity of the NH 2 domain of LjPLP-IV was confirmed by assaying directly the transferase activity for phosphatidylinositol and phosphatidylcholine in recombinant protein expressed in Escherichia coli . The authors propose that the bidirectional promoter producing antisense provides a mechanism for controlling expression of the LjPLP-IV and the autoinhibitory COOH-terminus provides yet another posttranslational mechanism for regulating PITP activity. This regulation of PITP is most likely important, because PITP activity can control the levels of phosphatidylinositol available for enzymes involved in producing second messenger signaling molecules from this lipid. P. Kapranov, S. M. Routt, V. A Bankaitis, F. J. de Bruijn, K. Szczyglowski, Nodule-specific regulation of phosphatidylinositol transfer protein expression in Lotus japonicus . Plant Cell 13 , 1369-1382 (2001). [Abstract] [Full Text]

Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2.

Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently. Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs. Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes. An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays. Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases. This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants. Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress.

Inositol lipids as spatial regulators of membrane traffic.

Phosphoinositides have been in the limelight for over two decades following their identification as sources of the second messengers, diacylglycerol, inositol(1,4,5) trisphosphate (IP3) and phosphatidylinositol(3,4,5) trisphosphate (PtdIns(3,4,5)P3). The last 10 years have witnessed an explosion of experimental results that demonstrate that inositol lipids themselves should be considered as second messengers in their own right. The intact lipid molecule functions as a reversible recruiting device for proteins to transiently bind to membranes and as an allosteric regulator of many proteins. The inositol headgroup of phosphatidylinositol (PtdIns) can be phosphorylated at a single or a combination of positions (38, 48 or 58) to give rise to seven different phosphoinositides (Fig. 1). Phosphoinositides can control membrane trafficking via their structural role as membrane components and via their ability to engage specific protein domains (e.g., pleckstrin homology (PH) or FVYE) which bind with high affinity and specificity to the different phosphorylated species of phosphoinositides, and thus recruit molecular machinery driving specific membrane trafficking events and regulating actin assembly. The first hint that phosphoinositides were important in membrane traffic came from studies showing that a bacterial phospholipase C inhibited regulated exocytosis in PC12 cells [20] and from genetic studies in Saccharomyces cerevisiae where the secretory mutant, Sec14 showed defects in exit of secretory vesicles from the Golgi and where the gene product responsible for these defects was identified as the yeast phosphatidylinositol transfer protein (PITP) [5]. Subsequent studies led to the identification of PITP, the small GTPase ARF, phosphatidylinositol 4-kinases (PI4K), phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and PIP phosphatases as regulators of membrane trafficking along the exocytic and endocytic pathways by virtue of their ability to control PtdIns(4,5)P2 levels [21, 30, 31, 34, 37, 44, 79]. As for 3-phosphorylated phosphoinositides, their role in membrane trafficking emerged with the identification of the product of the Vps34 gene (whose mutation was responsible for a defect in vacuolar protein sorting and membrane trafficking) as phosphoinositide 3-kinase (PI3K) which phosphorylates PtdIns to PtdIns(3)P [65, 66]. Since then many studies have provided evidence for a role of PI3Ks and their regulators, such as the small GTPase Rab5, in Golgi to endosomal/vacuole transport and along the endocytic pathway both in yeast and mammals and for the existence of a selective PI(3)P binding domain in some proteins, the FVYE domain [9, 25, 58]. Although the role of inositol lipids in membrane traffic appears to be central in yeast and in mammals, it is also clear that mammalian systems, due to their inherent complexity, have a richer and wider variety of functions where phosphoinositides are important. This is also apparent when one compares the inositol lipidmetabolizing enzymes and the inositol lipids identified in mammalian organisms and in S. cerevisiae (compare Fig. 1 and Fig. 2 and see also Table). PtdIns is the parent lipid from which all the phosphorylated species are derived (Fig. 1). In mammalian cells, PtdIns constitutes between 5–8% of total cellular lipids and the enzymatic Correspondence to: S. Cockcroft