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.     

Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2

Background

Two phenylalanines (FF) in an acidic tract (FFAT)-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA–E). Such motifs are found in several lipid transfer protein (LTP) families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP). Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors.

Results

We used a quantifiable in vivo system that measured ER targeting in a reporter yeast strain that over-expressed VAP to study the effect of substituting different elements of FFAT-like motifs in turn. By defining FFAT-like motifs more widely than before, we found them in novel proteins the functions of which had not previously been directly linked to the ER, including: two PKA anchoring proteins, AKAP220 and AKAP110; a family of plant LTPs; and the glycolipid LTP phosphatidylinositol-four-phosphate adaptor-protein-2 (FAPP-2).

Conclusion

All of the seven essential elements of a FFAT motif tolerate variation, and weak targeting to the ER via VAP is still detected if two elements are substituted. In addition to the strong FFAT motifs already known, there are additional proteins with weaker FFAT-like motifs, which might be functionally important VAP interactors.     

Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites

Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP‐A and VAP‐B, interact with proteins from other organelles that possess a small VAP‐interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain‐containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro. MOSPD2 is an ER‐anchored protein, and it interacts with several FFAT‐containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle‐bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.     

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.     

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.     

Phosphoregulation of the Ceramide Transport Protein CERT at Serine 315 in the Interaction with VAMP-associated Protein (VAP) for Inter-organelle Trafficking of Ceramide in Mammalian Cells

The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We show that the phosphorylation of CERT at serine 315, which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at Ser-315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine-rich motif) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the serine-rich motif and the phosphorylation of Ser-315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.     

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.     

FFAT motif phosphorylation controls formation and lipid transfer function of inter‐organelle contacts

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP‐A, VAP‐B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP‐A, VAP‐B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non‐conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non‐conventional FFAT motifs (named Phospho‐FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho‐FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho‐FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER‐endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter‐organelle contacts.     

神经酰胺转运蛋白在非囊泡转运中的作用机制

神经酰胺转运蛋白（ceramide transfer protein,CERT）是介导神经酰胺（ceramide）非囊泡转运的载体.它包括3个功能区域： PH、FFAT和START.PH和FFAT分别发挥高尔基体和内质网的靶向作用,羧基端的START主要用于与神经酰胺结合.CERT的转运受多种因素的调节,依赖于PKD和PP2Cε诱导的丝氨酸重复区域（SR）的磷酸化和去磷酸化,氧化应激刺激的CERT三聚体形成,以及PI4KⅢβ催化的高尔基体接头PI4P的生成等.CERT功能障碍会导致细胞易受氧化应激的损害.本文拟从CERT的结构、作用及其调节机制3方面进行综述,揭示CERT的研究进展.     

CERT-mediated trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT mediates the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains and motifs including i) a START domain capable of catalyzing inter-membrane transfer of ceramide, ii) a pleckstrin homology domain, which serves to target the Golgi apparatus, iii) a FFAT motif which interacts with the ER-resident membrane protein VAP, and iv) a serine-repeat motif, of which hyperphosphorylation down-regulates CERT activity. It has been suggested that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that efficient CERT-mediated trafficking of ceramide occurs at membrane contact sites between the ER and the Golgi apparatus.     

Molecular mechanisms and regulation of ceramide transport

De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi-ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.     

CERT and intracellular trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT has been identified as a key factor for the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains including (i) a START domain capable of catalyzing inter-membrane transfer of ceramide, (ii) a pleckstrin homology domain, which serves to target the Golgi apparatus by recognizing phosphatidylinositol 4-monophosphate, and (iii) a short peptide motif named FFAT motif which interacts with the ER-resident membrane protein VAP. CERT is preferentially distributed to the Golgi region in cells, and Golgi-targeted CERT appears to retain the activity to interact with VAP. On the basis of these results, it has been proposed that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that a particularly efficient cycle of CERT movement for trafficking of ceramide may proceed at membrane contact sites between the ER and the Golgi apparatus.     

The C-terminal dilysine motif confers endoplasmic reticulum localization to type I membrane proteins in plants

The tomato Cf-9 disease resistance gene encodes a type I membrane protein carrying a cytosolic dilysine motif. In mammals and yeast, this motif promotes the retrieval of type I membrane proteins from the Golgi apparatus to the endoplasmic reticulum (ER). To test whether the C-terminal KKXX signal of Cf-9 is functional as a retrieval motif and to investigate its role in plants, green fluorescent protein (GFP) was fused to the transmembrane domain of Cf-9 and expressed in yeast, Arabidopsis, and tobacco cells. The fusion protein was targeted to the ER in each of these expression systems, and mutation of the KKXX motif to NNXX led to secretion of the fusion protein. In yeast, the mutant protein reached the vacuole, but plants secreted it as a soluble protein after proteolytic removal of the transmembrane domain. Triple hemagglutinin (HA)-tagged full-length Cf-9 was also targeted to the ER in tobacco cells, and cleavage was also observed for the NNXX mutant protein, suggesting an endoprotease recognition site located within the Cf-9 lumenal sequence common to both the GFP- and the HA-tagged fusions. Our results indicate that the KKXX motif confers ER localization in plants as well as mammals and yeast and that Cf-9 is a resident protein of the ER.     

Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A–binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein–associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome–ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB—and thus peroxisome–ER contact sites—differently. Moreover, we demonstrate that GSK3β (glycogen synthase kinase-3 β) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome–ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.     

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 molecular mechanisms that underlie neurite formation include both cytoskeletal remodeling and membrane trafficking. Membrane components are transported in a directional manner within the cell by a membrane recycling system, resulting in expansion of the surface area of the neurite. The small GTPase Rab11 regulates membrane recycling and constitutive exocytosis (1), and it is thought to contribute to neurite formation through regulation of directional membrane transport.We have recently identified protrudin as a key regulator of Rab11-dependent membrane trafficking during neurite extension. Protrudin interacts with FKBP38 (also known as FKBP8) (2), which is a member of the immunophilin family of proteins that bind the immunosuppressant drug FK506 (3). FKBPs are multifunctional proteins that regulate the folding or export of other proteins as a result of their peptidyl-prolyl cis-trans-isomerase activity (4). Protrudin was found to interact with FKBP38, but not with other FKBP proteins such as FKBP12 or FKBP52 (5). Protrudin is hyperphosphorylated in Fkbp38^-/- mice, which manifest abnormal extension of nerve fibers (5).Protrudin contains a Rab11-binding domain (RBD11), two transmembrane domains (TM1 and TM2),² an FFAT (two phenylalanines in an acidic tract) motif (6), a coiled-coil domain, and a FYVE domain (7). These structural characteristics suggested that protrudin might function in membrane trafficking, particularly in membrane recycling. The gene encoding ZFYVE27 (a synonym of human protrudin) was recently found to be mutated in a German family with an autosomal dominant form of hereditary spastic paraplegia (AD-HSP), which is characterized by selective degeneration of axons (8). The phenotype of the affected individuals is similar to that of patients with AD-HSP caused by mutation of spastin, a protein implicated in neuronal vesicular trafficking (9), and protrudin was shown to interact with spastin (8). These findings support the notion that protrudin plays a key role in Rab11-mediated directional membrane transport during neurite formation.The subcellular localization of protrudin is dynamic. Whereas it is localized to the endoplasmic reticulum (ER) under basal conditions, nerve growth factor (NGF) triggers the translocation of protrudin from the ER, via recycling endosomes, to the tip of membrane protrusions in neuronal cells. Given that the FFAT motif is thought to serve as an ER targeting signal (6), this motif might be expected to contribute both to the localization of protrudin to the ER and to the regulation of neurite formation by this protein. The FFAT motif (consensus amino acid sequence of EFFDAXE, where X is any amino acid) is present in several lipid-binding proteins that are implicated in the transfer of lipids between the ER and other organelles such as the Golgi apparatus (10, 11). Vesicle-associated membrane protein-associated protein (VAP) interacts with these lipid-binding proteins through their FFAT motifs (6, 11, 12). The VAP-A and VAP-B isoforms of mammalian VAP are ER-resident type II membrane proteins (13) that are encoded by different genes (14); VAP-C is a splicing variant of VAP-B that lacks the membrane-spanning domain. VAP-A and VAP-B share ∼60% amino acid sequence identity, form homo- or heterodimers, and are expressed in many tissues (14-16). In addition to their localization to the ER (16), VAP-A and VAP-B are present in a wide range of intracellular membranes or membrane structures, including the Golgi, the ER-Golgi intermediate compartment (17), tight junctions (18), neuromuscular junctions (19), recycling endosomes, and the plasma membrane (20).We have now identified VAP-A and VAP-B as proteins that interact with protrudin. Protrudin preferentially interacts with VAP-A via its FFAT motif, and this motif was found to be required for the protrudin-dependent formation of membrane protrusions in HeLa cells. In addition, depletion of VAP-A by RNA interference resulted in inhibition of NGF-induced neurite outgrowth in the PC12 rat pheochromocytoma cell line. This inhibition of neurite outgrowth was reversed by expression of human VAP-A but not by that of VAP-A mutants that have lost the ability to bind to protrudin. These results suggest that interaction of protrudin with VAP-A is important both for its ER retention and for its ability to stimulate neurite formation.     

Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins

Membrane proteins destined for the vacuolar or lysosomal lumen are typically ubiquitinated, the ubiquitin serving as a targeting signal for the multivesicular body pathway. The RING-domain ubiquitin ligase Tul1 is an integral membrane protein that modifies the yeast vacuolar enzyme carboxypeptidase S (Cps1), the polyphosphatase Ppn1/Phm5 and other proteins containing exposed hydrophilic residues within their transmembrane domains (TMDs). Here we show that Bsd2 provides an alternative ubiquitination mechanism for Cps1, Phm5 and other proteins. Bsd2 is a three-TMD protein with a PPXY motif that binds the HECT domain ubiquitin ligase Rsp5. It can thus act as a specific adaptor linking Rsp5 to its substrates. Like Tul1, the Bsd2 system recognises polar TMDs. Bsd2 also controls the vacuolar targeting of a manganese transporter and a mutant plasma membrane ATPase, and together with the ER retrieval receptor Rer1, it protects cells from stress. We suggest that Bsd2 has a wide role in the quality control of membrane proteins. Bsd2 is the yeast homologue of human NEDD4 binding protein N4WBP5, which may therefore have similar functions.     

Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.     

Role of ORPs in sterol transport from plasma membrane to ER and lipid droplets in mammalian cells

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.