Phosphatidylinositol transfer proteins couple lipid transport to phosphoinositide synthesis

Phosphatidylinositol transfer proteins (PITPs) are lipid binding proteins that can catalyse the transfer of phosphatidylinositol (PI) from membranes enriched in PI to PI-deficient membranes. Three soluble forms of PITP of 35--38 kDa (PITP alpha, PITP beta and rdgB beta) and two larger integral proteins of 160 kDa (rdgB alpha I and II), which contain a PITP domain, are found in mammalian cells. PITPs are intimately associated with the compartmentalised synthesis of different phosphorylated inositol lipids. PI is the primary inositol lipid that is synthesised at the endoplasmic reticulum and is further phosphorylated in distinct membrane compartments by many specific lipid kinases to generate seven phosphorylated inositol lipids which are required for both signalling and for membrane traffic. PITPs play essential roles in both signalling via phospholipase C and phosphoinositide 3-kinases and in multiple aspects of membrane traffic including regulated exocytosis and vesicle biogenesis.     

RdgB proteins: functions in lipid homeostasis and signal transduction

The RdgBs are a group of evolutionarily conserved molecules that contain a phosphatidylinositol transfer protein (PITP) domain. However in contrast to classical PITPs (PITPalpha) with whom they share the conserved PITP domain, these proteins also contain several additional sequence elements whose functional significance remains unknown. The founding member of the family DrdgB alpha (Drosophila rdgB) appears to be essential for sensory transduction and maintenance of ultra structure in photoreceptors (retinal sensory neurons). Although proposed to support the maintenance of phosphatidylinositol 4, 5 bisphosphate [PI (4, 5) P(2)] levels during G-protein coupled phospholipase C activity in these cells, the biochemical mechanism of DrdgB alpha function remains unresolved. More recently, a mammalian RdgB protein has been implicated in the maintenance of diacylglycerol (DAG) levels and secretory function at Golgi membranes. In this review we discuss existing work on the function of RdgB proteins and set out future challenges in understanding this group of lipid transfer proteins.     

Phosphatidylinositol transfer proteins: a requirement in signal transduction and vesicle traffic

Phosphatidylinositol transfer protein (PITP) was originally identified and named because of its ability to transport phosphatidylinositol through the aqueous phase from one membrane compartment to another. Recent data, however, indicate unanticipated roles for PITP in the coupling of PIP₂ synthesis to signal transduction reactions and to membrane traffic in mammalian cells. PITP was recently purified on the basis of its ability to restore cellular functions in permeabilized cells depleted of cytosolic proteins. These functions include cell-surface receptor-regulated hydrolysis of PIP₂ by phospholipases Cβ- and γ-isozymes, regulated release of secretory granules, and the budding of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network. In the yeast Saccharomyces cerevisiae, a PITP was identified from a mutant strain with a defect in the secretory pathway (SEC14) and therefore required for cell viability; in Yarrowia lipolytica, PITP is required for differentiation from a yeast to a mycelial growth form. We are just beginning to unravel the intriguing mechanisms by which PITP/SEC14 may accomplish its function in eukaryotic cells in signal transduction and membrane trafficking. BioEssays 20 :423-432, 1998. © 1998 John Wiley & Sons Inc.     

Mammalian phosphatidylinositol transfer proteins: emerging roles in signal transduction and vesicular traffic.

Phosphatidylinositol transfer proteins (PITP) are abundant cytosolic proteins found in all mammalian cells. Two cytosolic isoforms of 35 and 36 kDa (PITP alpha and PITP beta) have been identified which share 77% identity. These proteins are characterized by having a single phospholipid binding site which exhibits dual headgroup specificity. The preferred lipid that can occupy the site can be either phosphatidylinositol (PI) or phosphatidylcholine (PC). In addition, PITP beta can also bind sphingomyelin. A second characteristic of these proteins is the ability to transfer PI and PC (or SM) from one membrane compartment to another in vitro. The function of PITP in mammalian cells has been examined mainly using reconstitution studies utilizing semi-intact cells or cell-free systems. From such analyses, a requirement for PITP has been identified in phospholipase C-mediated phosphatidylinositol bisphosphate (PI(4,5)P2) hydrolysis, in phosphoinositide 3-kinase catalyzed PIP3 generation, in regulated exocytosis, in the biogenesis of secretory granules and vesicles and in intra-golgi transport. Studies aimed at elucidating the mechanism of action of PITP in each of these seemingly disparate processes have yielded a singular theme: the activity of PITP stems from its ability to transfer PI from its site of synthesis to sites of cellular activity. This function was predicted from its in vitro characteristics. The second feature of PITP that was not predicted is the ability to stimulate the local synthesis of several phosphorylated forms of PI including PI(4)P, PI(4,5)P2, PI(3)P, PI(3,4,5)P3 by presenting PI to the lipid kinases involved in phosphoinositide synthesis. We conclude that PITP contributes in multiple aspects of cell biology ranging from signal transduction to membrane trafficking events where a central role for phosphoinositides is recognized either as a substrate or as an intact lipid signalling molecule.     

Secretogranin III directs secretory vesicle biogenesis in mast cells in a manner dependent upon interaction with chromogranin A

Mast cells are granular immunocytes that reside in the body's barrier tissues. These cells orchestrate inflammatory responses. Proinflammatory mediators are stored in granular structures within the mast cell cytosol. Control of mast cell granule exocytosis is a major therapeutic goal for allergic and inflammatory diseases. However, the proteins that control granule biogenesis and abundance in mast cells have not been elucidated. In neuroendocrine cells, whose dense core granules are strikingly similar to mast cell granules, granin proteins regulate granulogenesis. Our studies suggest that the Secretogranin III (SgIII) protein is involved in secretory granule biogenesis in mast cells. SgIII is abundant in mast cells, and is organized into vesicular structures. Our results show that over-expression of SgIII in mast cells is sufficient to cause an expansion of a granular compartment in these cells. These novel granules store inflammatory mediators that are released in response to physiological stimuli, indicating that they function as bona fide secretory vesicles. In mast cells, as in neuroendocrine cells, we show that SgIII is complexed with Chromogranin A (CgA). CgA is granulogenic when complexed with SgIII. Our data show that a novel non-granulogenic truncation mutant of SgIII (1-210) lacks the ability to interact with CgA. Thus, in mast cells, a CgA-SgIII complex may play a key role in secretory granule biogenesis. SgIII function in mast cells is unlikely to be limited to its partnership with CgA, as our interaction trap analysis suggests that SgIII has multiple binding partners, including the mast cell ion channel TRPA1.     

A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components

In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.     

Current thoughts on the phosphatidylinositol transfer protein family

Monomeric transport of lipids is carried out by a class of proteins that can shield a lipid from the aqueous environment by binding the lipid in a hydrophobic cavity. One such group of proteins is the phosphatidylinositol transfer proteins (PITP) that can bind phosphatidylinositol and phosphatidylcholine and transfer them from one membrane compartment to another. PITPs are found in both unicellular and multicellular organisms but not bacteria. In mice and humans, the PITP domain responsible for lipid transfer is found in five proteins, which can be classified into two classes based on sequence. Class I PITPs comprises two family members, alpha and beta, small 35 kDa proteins with a single PITP domain which are ubiquitously expressed. Class IIA PITPs (RdgBalphaI and II) are larger proteins possessing additional domains that target the protein to membranes and are only able to bind lipids but not mediate transfer. Finally, Class IIB PITP (RdgBbeta) is similar to Class I in size (38 kDa) and is also ubiquitously expressed. Class III PITPs, exemplified by the Sec14p family, are found in yeast and plants but are unrelated in sequence and structure to Class I and Class II PITPs. In this review we discuss whether PITP proteins are passive transporters or are regulated proteins that are able to couple their transport and binding properties to specific biological functions including inositol lipid signalling and membrane turnover.     

Mice lacking phosphatidylinositol transfer protein-alpha exhibit spinocerebellar degeneration,intestinal and hepatic steatosis,and hypoglycemia

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions. We now report that ablation of PITP alpha function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice. The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects. The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum. We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITP alpha-/- mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease. Taken together, the data suggest an unanticipated role for PITP alpha in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes.     

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.     

Tryptase precursors are preferentially and spontaneously released,whereas mature tryptase is retained by HMC-1 cells,Mono-Mac-6 cells,and human skin-derived mast cells

Tryptase (alpha and beta) levels in serum are used to assess mast cell involvement in human disease. Using cultured cells, the current study examines the hypothesis that protryptase(s) are spontaneously secreted by mast cells at rest, whereas mature tryptase(s) are stored in secretory granules until their release by activated cells. HMC-1 cells have only beta-tryptase genes and the corresponding mRNA. Mono-Mac-6 cells have both alpha- and beta-tryptase genes but preferentially express alpha-tryptase. Mono-Mac-6 cells spontaneously secrete most of their tryptase, which consists of alpha-protryptase, whereas mature tryptase is retained inside these cells. HMC-1 cells also spontaneously secrete most of their tryptase, identified as beta-protryptase, and retain mature tryptase. Skin-derived mast cells retain most of their tryptase, which is mature, and spontaneously secrete protryptase(s). Total tryptase levels in plasma are detectable but no different in healthy subjects with and without the gene for alpha-tryptase, consistent with pro forms of both alpha- and beta-tryptase being spontaneously secreted. Thus, protryptase(s) are spontaneously secreted by resting mast cells, whereas mature tryptase is retained by mast cells until they are activated to degranulate.     

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.     

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.     

Phosphatidylinositol/phosphatidylcholine transfer proteins in yeast

Phosphatidylinositol transfer proteins (PITPs) are now becoming widely recognized as intriguing proteins that participate in the coordination and coupling of phospholipid metabolism with vesicle trafficking, and in the regulation of important signaling cascades. Yet, only in one case is there a large body of evidence that speaks to the precise identities of PITP-dependent cellular reactions, and to the mechanisms by which PITPs execute function in eukaryotic cells. At present, yeast provide the most powerful system for analysis of the physiology of PITP function in vivo, and the mechanism by which this function is carried out. Here, we review the recent progress and remaining questions in the area of PITP function in yeast.     

In vitro lipid transfer assays of phosphatidylinositol transfer proteins provide insight into the in vivo mechanism of ligand transfer

Fluorescence resonance energy transfer (FRET) assays and membrane binding determinations were performed using three phosphatidylinositol transfer proteins, including the yeast Sec14 and two mammalian proteins PITPα and PITPβ. These proteins were able to specifically bind the fluorescent phosphatidylcholine analogue NBD-PC ((2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine)) and to transfer it to small unilamellar vesicles (SUVs). Rate constants for transfer to vesicles comprising 100% PC were slower for all proteins than when increasing percentages of phosphatidylinositol were incorporated into the same SUVs. The rates of ligand transfer by Sec14 were insensitive to the inclusion of equimolar amounts of another anionic phospholipid phosphatidylserine (PS), but the rates of ligand transfer by both mammalian PITPs were strikingly enhanced by the inclusion of phosphatidic acid (PA) in the receptor SUV. Binding of Sec14 to immobilized bilayers was substantial, while that of PITPα and PITPβ was 3–7 times weaker than Sec14 depending on phospholipid composition. When small proportions of the phosphoinositide PI(4)P were included in receptor SUVs (either with PI or not), Sec14 showed substantially increased rates of NBD-PC pick-up, whereas the PITPs were unaffected. The data are supportive of a role for PITPβ as functional PI transfer protein in vivo, but that Sec14 likely has a more elaborate function.     

An immunocytochemical study of the endocrine pancreas in the Australian fat-tailed dunnart (Sminthopsis crassicaudata)

Summary The endocrine pancreas of the Australian fattailed dunnart, Sminthopsis crassicaudata, was investigated by means of electron-microscopic immunocytochemistry using the protein A-gold technique on London resin (LR) white-embedded tissue. The primary antibodies used were raised against insulin, glucagon, somatostatin and pancreatic polypeptide. The morphology of the secretory granules differed in the four cell types. The insulin cells are pleomorphic, and the secretory granules composed of an electron-dense core surrounded by an electron-lucen halo. The glucago cells possess granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells have large, less dense secretory granules. The pancreatic polypeptide cells show small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborated by immunocytochemical data at the light-or preferably electron-microscopic level. Recent developments in immuno-electron-microscopic techniques have contributed to a better knowledge of cells responsible for the secretion of a wide variety of hormones, as in this study.     

Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.     

Immortalization of murine connective tissue-type mast cells at multiple stages of their differentiation by coculture of splenocytes with fibroblasts that produce Kirsten sarcoma virus

Mature connective tissue mast cells (CTMC) have not been previously available as a cell line from any species. Here we describe 15 novel mast cell lines (KiSV-MC) that were derived by coculturing murine splenocytes with fibroblasts that produce a Ki-ras-containing murine sarcoma virus. Some of the KiSV-MC lines are similar to CTMC in that they synthesize predominantly heparin proteoglycans, and contain up to 35 micrograms of histamine and 2.2 units of carboxypeptidase A/10(6) cells in secretory granules which stain red with Safranin. Other cell lines display phenotypic characteristics intermediate to CTMC and mucosal-like mast cells in being predominantly Safranin-, having lower amounts of histamine and carboxypeptidase A, and in synthesizing chondroitin sulfate E proteoglycans in preference to heparin proteoglycans. When the individual KiSV-MC lines were compared, a linear relationship was found between the number of Safranin+ granules, the cellular contents of histamine and carboxypeptidase A, and the biosynthesis of heparin relative to chondroitin sulfate E proteoglycans. Upon sensitization with monoclonal IgE and exposure to hapten-specific antigen, the cells exocytose the contents of their secretory granules. Thus, these immortalized cells provide the first source of CTMC-like lines for chemical and functional analysis and illustrate that murine mast cells can express a continuum of phenotypes.     

Constitutive and basal secretion from the endocrine cell line, AtT-20

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.