FFAT Motif Research Articles

Sterol binding by OSBP-related protein 1L regulates late endosome motility and function.

ORP1L is an oxysterol binding homologue that regulates late endosome (LE) positioning. We show that ORP1L binds several oxysterols and cholesterol, and characterize a mutant, ORP1L Δ560-563, defective in oxysterol binding. While wild-type ORP1L clusters LE, ORP1L Δ560-563 induces LE scattering, which is reversed by disruption of the endoplasmic reticulum (ER) targeting FFAT motif, suggesting that it is due to enhanced LE-ER interactions. Endosome motility is reduced upon overexpression of ORP1L. Both wild-type ORP1L and the Δ560-563 mutant induce the recruitment of both dynactin and kinesin-2 on LE. Most of the LE decorated by overexpressed ORP1L fail to accept endocytosed dextran or EGF, and the transfected cells display defective degradation of internalized EGF. ORP1L silencing in macrophage foam cells enhances endosome motility and results in inhibition of [(3)H]cholesterol efflux to apolipoprotein A-I. These data demonstrate that LE motility and functions in both protein and lipid transport are regulated by ORP1L.

Electrostatic Interaction between Oxysterol-binding Protein and VAMP-associated Protein A Revealed by NMR and Mutagenesis Studies

Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-A(MSP)) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBP(F)) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-A(MSP), OSBP(F), and the complex. Our results show that OSBP(F) is disordered in the free state, and VAP-A(MSP) and OSBP(F) form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain.

Structural Requirements for VAP-B Oligomerization and Their Implication in Amyotrophic Lateral Sclerosis-associated VAP-B(P56S) Neurotoxicity

The integral endoplasmic reticulum (ER)-membrane protein VAP-B interacts with various lipid-transfer/binding proteins containing an FFAT motif through its N-terminal MSP domain. A genetic mutation within its MSP domain, P56S, was identified in familial forms of motor neuron diseases. This mutation induces the formation of insoluble VAP-B(P56S) protein aggregates by an unknown mechanism. In this study, we defined the structural requirements for VAP-B oligomerization and demonstrated their contribution for VAP-B(P56S) aggregation and neurotoxicity. We show that the oligomerization of VAP-B is mainly mediated by its coiled-coil domain and that the GXXXG dimerization motif within the transmembrane domain mediates transmembrane domains self-association but is insufficient to drive VAP-B oligomerization. We further show that the oligomerization of the wild-type VAP-B is independent of its MSP domain. However, we found that the P56S mutation induces conformational changes within the MSP domain and facilitates its propensity to aggregate by exposing hydrophobic patches to the solvent. These conformational changes have no direct effect on FFAT binding. Rather, they enhance VAP-B(P56S) oligomerization driven by the combined contributions of the coiled-coil and the transmembrane domains, thereby preventing accessibility to FFAT-binding site, facilitating the production of VAP-B(P56S)-insoluble aggregates and consequently its neurotoxicity. These results shed light on the mechanism by which VAP-B(P56S) aggregates are formed and induce familial motor neuron diseases.

Role of the transcription factor Opi1p and phosphatidic acid in the formation of lipid droplets in Saccharomyces cerevisiae

Phosphatidic acid (PA) is a signaling molecule that plays an important role in mediating yeast phospholipid biosynthesis. In cells grown in the absence of inositol, the transcriptional repressor Opi1p is localized to the endoplasmic reticulum (ER) through interactions with Scs2p via its FFAT motif (two phenylalanines in an acidic tract) and the binding with PA. Under these conditions, the UASINO‐containing phospholipid biosynthetic genes are expressed at the maximum level through activation via the Ino2p‐Ino4p activation complex. When inositol is added to the medium, the increased synthesis of phosphatidylinositol causes a rapid depletion of PA in the ER. Opi1p then translocates to the nucleus and represses the transcription of target genes (Loewen et al. 2004. Science 304, 1644–1647). Here we present studies utilizing Opi1p reporter constructs to monitor the change in PA metabolism. After the cells grew under inositol starvation for 2 hrs, novel punctate structures were recognized by Opi1p‐GFP in the FFAT and scs2 null mutants. Interestingly, these structures are highly associated with ER. Furthermore, by using Tgl4p‐RFP as a lipid droplet marker to identify these structures, we observed colocalization of Tgl4p‐RFP with the punctate structures. These results suggest a role for both Opi1p and PA in the formation of lipid droplets.Supported by NIH grant GM019629.

The glycolipid transfer protein interacts with the vesicle-associated membrane protein-associated protein VAP-A

The glycolipid transfer protein (GLTP) is a cytoplasmic protein with an ability to bind glycolipids and catalyze their in vitro transfer. In this study, we have found a FFAT-like motif in GLTP. The FFAT (two phenylalanines in an acidic tract) motif in lipid-binding proteins has previously been shown to interact with the VAPs (vesicle-associated membrane protein-associated proteins) in the endoplasmic reticulum. Here we used glutathione S-transferase pull-down experiments to confirm that GLTP and VAP-A interact. By displacing different amino acids in the motif we clearly show that the interaction is dependent on the FFAT-like motif in GLTP. The potential role of GLTP in the endoplasmic reticulum association is discussed.

Promotion of Neurite Extension by Protrudin Requires Its Interaction with Vesicle-associated Membrane Protein-associated Protein

Protrudin is a protein that contains a Rab11-binding domain and a FYVE (lipid-binding) domain and that functions to promote neurite formation through interaction with the GDP-bound form of Rab11. Protrudin also contains a short sequence motif designated FFAT (two phenylalanines in an acidic tract), which in other proteins has been shown to mediate binding to vesicle-associated membrane protein-associated protein (VAP). We now show that protrudin associates and colocalizes with VAP-A, an isoform of VAP expressed in the endoplasmic reticulum. Both the interaction between protrudin and VAP-A as well as the induction of process formation by protrudin were markedly inhibited by mutation of the FFAT motif. Furthermore, depletion of VAP-A by RNA interference resulted in mislocalization of protrudin as well as in inhibition of neurite outgrowth induced by nerve growth factor in rat pheochromocytoma PC12 cells. These defects resulting from depletion of endogenous rat VAP-A in PC12 cells were corrected by forced expression of (RNA interference-resistant) human VAP-A but not by VAP-A mutants that have lost the ability to interact with protrudin. These results suggest that VAP-A is an important regulator both of the subcellular localization of protrudin and of its ability to stimulate neurite outgrowth.

The influence of phospholipid metabolism and regulatory networks on the subcellular localization of phosphatidic acid in Saccharomyces cerevisiae

Phosphatidic acid (PA) is a signaling molecule that plays an important role in mediating yeast phospholipid biosynthesis. In cells grown in the absence of inositol, the transcriptional repressor Opi1p is localized to the endoplasmic reticulum (ER) through interactions with Scs2p via its FFAT motif (two phenylalanines in an acidic tract) and the binding with PA. Under these conditions, the UASINO‐containing phospholipid biosynthetic genes are expressed at the maximum level through activation via the Ino2p‐Ino4p activation complex. When inositol is added to the medium, the increased synthesis of phosphatidylinositol causes a rapid depletion of PA in the ER. Opi1p then translocates to the nucleus and represses the transcription of target genes (Loewen et al. 2004. Science 304, 1644 ‐ 1647). Here we present studies utilizing Opi1p reporter constructs to monitor the change in PA metabolism. Strains carrying a single genomic copy of a wild type OPI1 or a mutant OPI1FFAT fused to GFP driven by the native OPI1 promoter were constructed. These functional constructs were introduced into strains contain mutations known to perturb PA metabolism or Opi1p localization including sec14ts, scs2Δ, single mutations or in combination with pct1Δ. INO1 expression was examined to compare with Opi1p localization results and to monitor Opi1p repressor activity under changing metabolic conditions.

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.

FFAT rescues VAPA-mediated inhibition of ER-to-Golgi transport and VAPB-mediated ER aggregation

The VAMP-associated proteins termed VAP are a small gene family of proteins characterised by the presence of an N-terminal major sperm protein (MSP) domain. The P56S mutation of the B isoform (VAPB) has been linked to late-onset amyotrophic lateral sclerosis (ALS8) and its expression causes formation of large ER aggregates. Overexpression of the wild-type A isoform (VAPA) but not the B isoform (VAPB), inhibited ER-to-Golgi transport of membrane proteins. This transport block by VAPA was primarily due to decreased segregation of membrane cargo into ER vesicles. We also found that VAPA inhibited lateral diffusion of membrane proteins, most likely through its stable association with microtubules. The MSP domain of VAP is known to interact with the FFAT motif (two phenylalanines in an acidic tract) of proteins involved in sterol regulation. Overexpression of FFAT restored ER-to-Golgi transport and lateral diffusion of membrane proteins, and resolved the large ER aggregates in VAPB-P56S. Application of a FFAT peptide restored in vitro ER vesicle budding and disrupted VAP-microtubule association. Thus, overexpression of the two VAP isoforms causes retention of ER membrane proteins by impeding lateral diffusion and their incorporation into transport vesicles. This inhibitory effect can be relieved by expression of the FFAT motif.

Coordinated Lipid Transfer between the Endoplasmic Reticulum and the Golgi Complex Requires the VAP Proteins and Is Essential for Golgi-mediated Transport

Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgi-mediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterol-binding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities.

Goodpasture Antigen-binding Protein and Its Spliced Variant, Ceramide Transfer Protein, Have Different Functions in the Modulation of Apoptosis during Zebrafish Development

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the alpha3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPDelta26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpDelta26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpDelta26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.

Two sphingolipid transfer proteins, CERT and FAPP2: Their roles in sphingolipid metabolism

Recent discoveries of two sphingolipid transfer proteins, CERT and FAPP2, have brought the field of sphingolipid metabolism to a more dynamic stage. CERT transfers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, a step crucial for sphingomyelin (SM) synthesis. The pleckstrin homology (PH) domain and the FFAT motif of CERT restrict the direction of transfer and destination of ceramide through binding to phosphatidylinositol 4-monophosphate (PI4P) at the Golgi and the ER resident proteins, VAPs, respectively. CERT is regulated by the phosphorylation and dephosphorylation of serine/threonine, in which protein kinase D, possibly casein kinase I, and PP2Cepsilon are involved. On the other hand, FAPP2 transfers glucosylceramide (GlcCer) to appropriate sites for the synthesis of complex glycosphingolipids. Like CERT, FAPP2 contains a PH domain, the binding of which to PI4P is required for its localization to the Golgi. These observations indicate that lipid transfer proteins, CERT and FAPP2, spatially regulate lipid metabolism on the cytosolic side.

The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity

The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition.

Motor Neuron Disease-Associated Mutant Vesicle-Associated Membrane Protein-Associated Protein (VAP) B Recruits Wild-Type VAPs into Endoplasmic Reticulum-Derived Tubular Aggregates

The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation (P56S) in the conserved major sperm protein homology domain of VAPB has been linked to motor-neuron degeneration in affected amyotrophic lateral sclerosis (ALS) patients. We report that in the CNS, VAPB is abundant in motor neurons and that the P56S substitution causes aggregation of mutant VAPB in immobile tubular ER clusters, perturbs FFAT-motif binding, and traps endogenous VAP in mutant aggregates. Expression of mutant VAPB or reduction of VAP by short hairpin RNA in primary neurons causes Golgi dispersion and cell death. VAPA and VAPB are reduced in human ALS patients and superoxide dismutase 1 (SOD1)-ALS-transgenic mice, suggesting that VAP family proteins may be involved in the pathogenesis of sporadic and SOD1-linked ALS. Our data support a model in which reduced levels of VAP family proteins result in decreased ER anchoring of lipid-binding proteins and cause motor neuron degeneration.

Interorganelle Trafficking of Ceramide Is Regulated by Phosphorylation-dependent Cooperativity between the PH and START Domains of CERT

The synthesis and transport of lipids are essential events for membrane biogenesis. However, little is known about how intracellular trafficking of lipids is regulated. Ceramide is synthesized at the endoplasmic reticulum (ER) and transported by the ceramide transfer protein CERT to the Golgi apparatus, where it is converted to sphingomyelin. CERT has a phosphoinositide-binding pleckstrin homology (PH) domain for Golgi-targeting and a lipid transfer START domain for intermembrane transfer of ceramide. We here show that CERT receives multiple phosphorylations at a serine-repeat motif, a possibe site for casein kinase I, and that the phosphorylation down-regulates the ER-to-Golgi transport of ceramide. In vitro assays show that the phosphorylation induces an autoinhibitory interaction between the PH and START domains and consequently inactivates both the phosphoinositide binding and ceramide transfer activities of CERT. Loss of sphingomyelin and cholesterol from cells causes dephosphorylation of CERT to activate it. The cooperative control of functionally distinct domains of CERT is a novel molecular event to regulate the intracellular trafficking of ceramide.

Efficient Trafficking of Ceramide from the Endoplasmic Reticulum to the Golgi Apparatus Requires a VAMP-associated Protein-interacting FFAT Motif of CERT

Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleck-strin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.

Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.

Structural Basis of FFAT Motif-Mediated ER Targeting

The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain. We have solved crystal structures of the rat VAP-A MSP homology domain alone and in complex with an FFAT motif. The co-crystal structure was used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant also blocked function of the VAP homolog Scs2p in yeast. Finally, overexpression of the FFAT binding-defective VAP in COS7 cells dramatically altered ER morphology. Our data establish the structural basis of FFAT-mediated ER targeting and suggest that FFAT-targeted proteins play an important role in determining ER morphology.

A Highly Conserved Binding Site in Vesicle-associated Membrane Protein-associated Protein (VAP) for the FFAT Motif of Lipid-binding Proteins

A variety of lipid-binding proteins contain a recently described motif, designated FFAT (two phenylalanines in an acidic tract), which binds to vesicle-associated-membrane protein-associated protein (VAP). VAP is a conserved integral membrane protein of the endoplasmic reticulum that contains at its amino terminus a domain related to the major sperm protein of nematode worms. Here we have studied the FFAT-VAP interaction in Saccharomyces cerevisiae, where the VAP homologue Scs2 regulates phospholipid metabolism via an interaction with the FFAT motif of Opi1. By introducing mutations at random into Scs2, we found that mutations that abrogated binding to FFAT were clustered in the most highly conserved region. Using site-directed mutagenesis, we identified several critical residues, including two lysines widely separated in the primary sequence. By examining all other conserved basic residues, we identified a third residue that was moderately important for binding FFAT. Modeling VAP on the known structure of major sperm protein showed that the critical residues form a patch on a positively charged face of the protein. In vivo functional studies of SCS22, a second SCS2-like gene in S. cerevisiae, showed that SCS2 was the dominant gene in the regulation of Opi1, with a minor contribution from SCS22. We then established that reduction in the affinity of Scs2 mutants for FFAT correlated well with loss of function, indicating the importance of these residues for binding FFAT motifs. Finally, we found that human VAP-A could substitute for Scs2 but that it functioned poorly, suggesting that other factors modulate the binding of Scs2 to proteins with FFAT motifs.

VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus

We recently showed that oxysterol-binding protein (OSBP), one of twelve related PH domain containing proteins with lipid and sterol binding activity, interacts with VAMP-associated protein (VAP)-A on the endoplasmic reticulum (ER). In addition to OSBP, seven OSBP-related proteins (ORPs) bind VAP-A via a conserved E-F/Y-F/Y-DA ‘FFAT’ motif. We focused on this interaction for ORP9, which is expressed as a full-length (ORP9L) or truncated version missing the PH domain (ORP9S). Mutation analysis showed that the interaction required the ORP9 FFAT motif and the N-terminal conserved domain of VAP. Endogenous ORP9L displayed Golgi localization, which was partially mediated by the PH domain based on limited localization of OPR9–PH–GFP with the Golgi apparatus. When inducibly overexpressed, ORP9S and ORP9L colocalized with VAP-A and caused vacuolation of the ER as well as retention of the ER–Golgi intermediate compartment marker ERGIC-53/p58 in the ER. ORP9L mutated in the VAP-A binding domain (ORP9L-FY→AA) did not localize to the ER but appeared with giantin and Sec31 on large vesicular structures, suggesting the presence of a hybrid Golgi–COPII compartment. Normal Golgi localization was also observed for ORP9L-FY→AA. Results show that VAP binding and PH domains target ORP9 to the ER and a Golgi–COPII compartment, respectively, and that ORP9L overexpression in these compartments severely perturbed their organization.