Localization of Drosophila retinal degeneration B, a membrane- associated phosphatidylinositol transfer protein

The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160- kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.     

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 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.     

Cloning and characterization of human homologue of Drosophila retinal degeneration B: A candidate gene for degenerative retinal diseases

Mutations in the Drosophila retinal degeneration B (D-rdgB) gene cause light-enhanced retinal degeneration. Here, we report the isolation of the cDNA encoding human homologue of the D-rdgB and initial characterization of the gene products. Like D-rdgB, the human rdgB homologue (H-rdgB) is a transmembrane protein with the N-terminus sharing high homology to two closely related cytosolic proteins, phosphatidylinositol transfer protein (PITP) α and β, indicating that rdgB like proteins belong to the family of PITP proteins. Using Northern and Western blotting, we demonstrated that the rdgB homologue is expressed in rat retina, olfactory bulb, and brain, but not in nonneuronal tissues. In the rat retina, immunoreactivity of the rdgB homologue was observed in photoreceptors and throughout the inner nuclear and plexiform layers; the strongest staining was in the inner plexiform layer. In the photoreceptor cells, the rdgB homologue was located primarily in the inner segment where sorting and traffic of membranes required for outer segment assembly take place. These data, together with recent findings showing PITPs as an important component of intracellular membrane traffic apparatus in mammalian cells, suggest that rdgB homologue may play a role in photoreceptor membrane renewal and in neurotransmitter release. Furthermore, using somatic hybrid cell hybridization and fluorescence in situ hybridization H-rdgB gene was mapped to human chromosome 11q13, a region known to contain several retinopathy loci, including Best disease and Bardet-Biedl syndrome I. Therefore, H-rdgB gene is an attractive candidate for several inherited retinal degenerative diseases. Dev. Genet. 20:235–245, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Involvement of PITPnm, a mammalian homologue of Drosophila rdgB, in phosphoinositide synthesis on Golgi membranes.

Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Commun. 236, 559-564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.     

Both isoforms of mammalian phosphatidylinositol transfer protein are capable of binding and transporting sphingomyelin

The structurally related mammalian α and β isoforms of phosphatidylinositol (PtdIns) transfer protein (PITP) bind reversibly a single phospholipid molecule, preferably PtdIns or phosphatidylcholine (PtdCho), and transport that lipid between membrane surfaces. PITPβ, but not PITPα, is reported extensively in the scientific literature to exhibit the additional capacity to bind and transport sphingomyelin (CerPCho). We undertook a detailed investigation of the lipid binding and transfer specificity of the soluble mammalian PITP isoforms. We employed a variety of donor and acceptor membrane lipid compositions to determine the sensitivity of recombinant rat PITPα and PITPβ isoforms toward PtdIns, PtdCho, CerPCho, and phosphatidate (PtdOH). Results indicated often striking differences in protein–phospholipid and protein–membrane interactions. We demonstrated unequivocally that both isoforms were capable of binding and transferring CerPCho; we confirmed that the β isoform was the more active. The order of transfer specific activity was similar for both isoforms: PtdIns>PtdCho>CerPCho≫PtdOH. Independently, we verified the binding of CerPCho to both isoforms by showing an increase in holoprotein isoelectric point following the exchange of protein-bound phosphatidylglycerol for membrane-associated CerPCho. We conclude that PITPα and PITPβ are able to bind and transport glycero- and sphingophospholipids.     

Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein

The protein tyrosine kinase PYK2 has been implicated in signaling pathways activated by G-protein-coupled receptors, intracellular calcium, and stress signals. Here we describe the molecular cloning and characterization of a novel family of PYK2-binding proteins designated Nirs (PYK2 N-terminal domain-interacting receptors). The three Nir proteins (Nir1, Nir2, and Nir3) bind to the amino-terminal domain of PYK2 via a conserved sequence motif located in the carboxy terminus. The primary structures of Nirs reveal six putative transmembrane domains, a region homologous to phosphatidylinositol (PI) transfer protein, and an acidic domain. The Nir proteins are the human homologues of the Drosophila retinal degeneration B protein (rdgB), a protein implicated in the visual transduction pathway in flies. We demonstrate that Nirs are calcium-binding proteins that exhibit PI transfer activity in vivo. Activation of PYK2 by agents that elevate intracellular calcium or by phorbol ester induce tyrosine phosphorylation of Nirs. Moreover, PYK2 and Nirs exhibit similar expression patterns in several regions of the brain and retina. In addition, PYK2-Nir complexes are detected in lysates prepared from cultured cells or from brain tissues. Finally, the Nir1-encoding gene is located at human chromosome 17p13.1, in proximity to a locus responsible for several human retinal diseases. We propose that the Nir and rdgB proteins represent a new family of evolutionarily conserved PYK2-binding proteins that play a role in the control of calcium and phosphoinositide metabolism downstream of G-protein-coupled receptors.     

Function of the phosphatidylinositol transfer protein gene family: is phosphatidylinositol transfer the mechanism of action?

Phosphatidylinositol transfer proteins (PITPs) bind and facilitate the transport of phosphatidylinositol (PI) and phosphatidylcholine between membrane compartments. They are highly conserved proteins, are found in both unicellular and multicellular organisms, and can be present as a single domain or as part of a larger, multi-domain protein. The hallmark of PITP proteins is their ability to sequester PI in their hydrophobic pocket. Ablation or knockdown of specific isoforms in vivo has wide ranging effects such as defects in signal transduction via phospholipase C and phosphoinositide 3-kinase, membrane trafficking, stem cell viability, Drosophila phototransduction, neurite outgrowth, and cytokinesis. In this review, we identify the common mechanism underlying each of these phenotypes as the cooperation between PITP proteins and lipid kinases through the provision of PI for phosphorylation. We propose that recruitment and concentration of PITP proteins at specific membrane sites are required for PITP proteins to execute their function rather than lipid transfer.     

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.     

Modifications of cysteine residues in the solution and membrane-associated conformations of phosphatidylinositol transfer protein have differential effects on lipid transfer activity

The alpha isoforms of mammalian phosphatidylinositol transfer protein (PITP) contain four conserved Cys residues. In this investigation, a series of thiol-modifying reagents, both alkylating and mixed disulfide-forming, was employed to define the accessibility of these residues and to evaluate their role in protein-mediated intermembrane phospholipid transport. Isolation and analysis of chemically modified peptides and site-directed mutagenesis of each Cys residue to Ala were also performed. Soluble, membrane-associated, and denatured preparations of wild-type and mutant rat PITPs were studied. Under denaturing conditions, all four Cys residues could be detected spectrophotometrically by chemical reaction with 4,4'-dipyridyl disulfide or 5,5'-dithiobis(2-nitrobenzoate). In the native protein, two of the four Cys residues were sensitive to some but not all thiol-modifying reagents, with discrimination based on the charge and hydrophobicity of the reagent and the conformation of the protein. With the soluble conformation of PITP, achieved in the absence of phospholipid vesicles, the surface-exposed Cys(188) was chemically modified without consequence to lipid transfer activity. Cys(188) exhibited an apparent pK(a) of 7.6. The buried Cys(95), which constitutes part of the phospholipid substrate binding site, was covalently modified upon transient association of PITP with a membrane surface. The Cys-to-Ala mutations showed that neither Cys(95) nor Cys(188) was essential for lipid transfer activity. However, chemical modification of Cys(95) resulted in the loss of lipid transfer activity. These results demonstrate that the Cys residues of PITP can be assigned to several different classes of chemical reactivity. Of particular interest is Cys(95), whose sulfhydryl group becomes exposed to modification in the membrane-associated conformation of PITP. Furthermore, the inhibition of PITP activity by thiol-modifying reagents is a result of steric hindrance of phospholipid substrate binding.     

14-3-3 protein and ATRAP bind to the soluble class IIB phosphatidylinositol transfer protein RdgBβ at distinct sites

PITPs (phosphatidylinositol transfer proteins) are characterized by the presence of the PITP domain whose biochemical properties of binding and transferring PI (phosphatidylinositol) are well studied. Despite their wide-spread expression in both unicellular and multicellular organisms, they remain functionally uncharacterized. An emerging theme is that individual PITPs play highly specific roles in either membrane trafficking or signal transduction. To identify specific roles for PITPs, identification of interacting molecules would shed light on their molecular function. In the present paper, we describe binding partners for the class IIB PITP RdgBβ (retinal degeneration type?Bβ). RdgBβ is a soluble PITP but is unique in that it contains a region of disorder at its C-terminus following its defining N-terminal PITP domain. The C-terminus of RdgBβ is phosphorylated at two serine residues, Ser274 and Ser299, which form a docking site for 14-3-3 proteins. Binding to 14-3-3 proteins protects RdgBβ from degradation that occurs at the proteasome after ubiquitination. In addition to binding 14-3-3, the PITP domain of RdgBβ interacts with the Ang II (angiotensin II)-associated protein ATRAP (Ang II receptor-associated protein). ATRAP is also an interacting partner for the AT1R (Ang II type?1 receptor). We present a model whereby RdgBβ functions by being recruited to the membrane by ATRAP and release of 14-3-3 from the C-terminus allows the disordered region to bind a second membrane to create a membrane bridge for lipid transfer, possibly under the control of Ang II.     

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.     

Endocytosis is involved in DNA uptake in yeast

The structurally related mammalian alpha and beta isoforms of phosphatidylinositol (PtdIns) transfer protein (PITP) bind reversibly a single phospholipid molecule, preferably PtdIns or phosphatidylcholine (PtdCho), and transport that lipid between membrane surfaces. PITPbeta, but not PITPalpha, is reported extensively in the scientific literature to exhibit the additional capacity to bind and transport sphingomyelin (CerPCho). We undertook a detailed investigation of the lipid binding and transfer specificity of the soluble mammalian PITP isoforms. We employed a variety of donor and acceptor membrane lipid compositions to determine the sensitivity of recombinant rat PITPalpha and PITPbeta isoforms toward PtdIns, PtdCho, CerPCho, and phosphatidate (PtdOH). Results indicated often striking differences in protein-phospholipid and protein-membrane interactions. We demonstrated unequivocally that both isoforms were capable of binding and transferring CerPCho; we confirmed that the beta isoform was the more active. The order of transfer specific activity was similar for both isoforms: PtdIns>PtdCho>CerPCho>PtdOH. Independently, we verified the binding of CerPCho to both isoforms by showing an increase in holoprotein isoelectric point following the exchange of protein-bound phosphatidylglycerol for membrane-associated CerPCho. We conclude that PITPalpha and PITPbeta are able to bind and transport glycero- and sphingophospholipids.     

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.     

A human protein specific for the immunoglobulin octamer DNA motif contains a functional homeobox domain

The homeobox domain is shared by Drosophila homeotic proteins, yeast mating type proteins, and some functionally uncharacterized mammalian proteins. A lymphoid-restricted human protein that binds to the immunoglobulin octamer regulatory motif was shown to contain an amino acid sequence that has 33% amino acid identity with the consensus sequence of the previously cloned homebox domains. This homeobox gene was localized to chromosome 19, thus mapping separately from other human homebox genes. A mutant protein containing amino acid substitutions within a putative helix-turn-helix motif in the homeobox domain did not bind DNA detectably. This human homeobox protein was shown to bind the same DNA sequence as the homeobox domains of the yeast mating type proteins and Drosophila homeotic protein, suggesting that homeobox proteins may have closely related DNA binding characteristics.     

The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein)

PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser(274) and Ser(299)) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.     

Intramembrane cleavage of ephrinB3 by the human rhomboid family protease, RHBDL2

Rhomboid-1 is a serine protease that cleaves the membrane domain of the Drosophila EGF-family protein, Spitz, to release a soluble growth factor. Several vertebrate rhomboid-like proteins have been identified, although their substrates and functions remain unknown. The human rhomboid, RHBDL2, cleaves the membrane domain of Drosophila Spitz when the proteins are co-expressed in mammalian cells. However, the membrane domains of several mammalian EGF-family proteins were not cleaved by RHBDL2, suggesting that the endogenous targets of the human protease are not EGF-related factors. We demonstrate that the amino acid sequence at the luminal face of the membrane domain of a substrate protein determines whether it is cleaved by RHBDL2. Based on this finding, we predicted B-type ephrins as potential RHBDL2 substrates. We found that one of these, ephrinB3, was cleaved so efficiently by the protease that little ephrinB3 was detected on the surface of cells co-expressing RHBDL2. These results raise the possibility that RHBDL2-mediated proteolytic processing may regulate intercellular interactions between ephrinB3 and eph receptors.