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.     

An Assembly of Proteins and Lipid Domains Regulates Transport of Phosphatidylserine to Phosphatidylserine Decarboxylase 2 in Saccharomyces cerevisiae

Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis of phosphatidylethanolamine. One route involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce phosphatidylethanolamine. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns 4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein-protein and protein-lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, which we name Pbi1p (PstB2p-interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER-localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N terminus of Psd2p (C2-1) as well the previously characterized C2 domain of Psd2p (C2-2). The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein-protein and protein-lipid interactions. Specifically, our model predicts that this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.     

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.     

Role for two conserved intermembrane space proteins, Ups1p and Up2p, in intra-mitochondrial phospholipid trafficking

Mitochondrial membranes maintain a specific phospholipid composition. Most phospholipids are synthesized in the endoplasmic reticulum (ER) and transported to mitochondria, but cardiolipin and phosphatidylethanolamine are produced in mitochondria. In the yeast Saccharomyces cerevisiae, phospholipid exchange between the ER and mitochondria relies on the ER-mitochondria encounter structure (ERMES) complex, which physically connects the ER and mitochondrial outer membrane. However, the proteins and mechanisms involved in phospholipid transport within mitochondria remain elusive. Here, we investigated the role of the conserved intermembrane space proteins, Ups1p and Ups2p, and an inner membrane protein, Mdm31p, in phospholipid metabolism. Our data show that loss of the ERMES complex, Ups1p, and Mdm31p causes similar defects in mitochondrial phospholipid metabolism, mitochondrial morphology, and cell growth. Defects in cells lacking the ERMES complex or Ups1p are suppressed by Mdm31p overexpression as well as additional loss of Ups2p, which antagonizes Ups1p. Combined loss of the ERMES complex and Ups1p exacerbates phospholipid defects. Finally, pulse-chase experiments using [(14)C]serine revealed that Ups1p and Ups2p antagonistically regulate conversion of phosphatidylethanolamine to phosphatidylcholine. Our results suggest that Ups proteins and Mdm31p play important roles in phospholipid biosynthesis in mitochondria. Ups proteins may function in phospholipid trafficking between the outer and inner mitochondrial membranes.     

Roles of SAM and DDHD domains in mammalian intracellular phospholipase A1 KIAA0725p

Members of the intracellular phospholipase A1 family of proteins have been implicated in organelle biogenesis and membrane trafficking. The mammalian family comprises three members: phosphatidic acid-preferring phospholipase A1 (PA-PIA1)/DDHD1, p125/Sec23ip and KIAA0725p/DDHD2, all of which have a DDHD domain. PA-PLAI is mostly cytosolic, while KIAA0725p and p125 are more stably associated with the Golgi/endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and ER exit sites, respectively. Here we show that KIAAO725p and p125 are novel phosphoinositide-binding proteins. Deletion and mutational analyses of KIAAO725p suggested that a sterile alpha-motif (SAM), which is also present inp125, but not in cytosolic PA-PLAI, and the following DDHD domain comprise a minimal region for phosphatidylinositol 4-phosphate (Pl(4)P)-binding. A construct with mutations in the positively charged cluster of the SAM domain is defective in both phosphoinositide-binding and Golgi/ERGIC targeting. Consistent with the view that the Pl(4)P-binding is important for the membrane association of KIAA0725p, expression of phosphoinositide phosphatase Sacd reduces the association of expressed KIAAO725p with membranes. In addition, we show that deletion of the DDHD domain or introduction of point mutations at the conserved aspartate or histidine residues in the domain abolishes the phospholipase activity of KIAAO725p and PA-PLA1. Together, our results suggest that KIAAO725p is targeted to specific organelle membranes in a phosphoinositide-dependent manner, and that its SAM and DDHD domains are essential for its phosphoinositide-binding and phospholipase activity.     

Der3p/Hrd1p Is Required for Endoplasmic Reticulum-associated Degradation of Misfolded Lumenal and Integral Membrane Proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3–1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61–2 allele. This is accompanied by the stabilization of the Sec61–2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61–2 strain at the permissive temperature of 25°C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61–2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.     

Genetic and structural analysis of Hmg2p-induced endoplasmic reticulum remodeling in Saccharomyces cerevisiae

The endoplasmic reticulum (ER) is highly plastic, and increased expression of distinct single ER-resident membrane proteins, such as HMG-CoA reductase (HMGR), can induce a dramatic restructuring of ER membranes into highly organized arrays. Studies on the ER-remodeling behavior of the two yeast HMGR isozymes, Hmg1p and Hmg2p, suggest that they could be mechanistically distinct. We examined the features of Hmg2p required to generate its characteristic structures, and we found that the molecular requirements are similar to those of Hmg1p. However, the structures generated by Hmg1p and Hmg2p have distinct cell biological features determined by the transmembrane regions of the proteins. In parallel, we conducted a genetic screen to identify HER genes (required for Hmg2p-induced ER Remodeling), further confirming that the mechanisms of membrane reorganization by these two proteins are distinct because most of the HER genes were required for Hmg2p but not Hmg1p-induced ER remodeling. One of the HER genes identified was PSD1, which encodes the phospholipid biosynthetic enzyme phosphatidylserine decarboxylase. This direct connection to phospholipid biosynthesis prompted a more detailed examination of the effects of Hmg2p on phospholipid mutants and composition. Our analysis revealed that overexpression of Hmg2p caused significant and specific growth defects in nulls of the methylation pathway for phosphatidylcholine biosynthesis that includes the Psd1p enzyme. Furthermore, increased expression of Hmg2p altered the composition of cellular phospholipids in a manner that implied a role for PSD1. These phospholipid effects, unlike Hmg2p-induced ER remodeling, required the enzymatic activity of Hmg2p. Together, our results indicate that, although related, Hmg2p- and Hmg1p-induced ER remodeling are mechanistically distinct.     

ER membrane protein complex required for nuclear fusion

Diploid cells of the yeast Saccharomyces cerevisiae form after the mating of two haploid cells of the opposite mating type. After fusion of the two plasma membranes of the mating cells, a dinucleated cell forms initially in which the two haploid nuclei then rapidly fuse to form a single diploid nucleus. This latter event, called karyogamy, can be divided into two distinct steps: the microtubule-based movement that causes the two nuclei to become closely juxtaposed and the fusion of the nuclear membranes. For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified. For topological reasons, however, it has been unclear how Kar2p could function in this role. Kar2p is localized to the luminal (i.e., noncytoplasmic) face of the ER membrane, yet nuclear fusion must initiate from the cytosolic side of the outer nuclear membrane or the ER membrane with which it is contiguous. There is both genetic and biochemical evidence that Kar2p interacts with Sec63p, an ER membrane protein containing both luminal and cytosolic domains that is involved in protein translocation across the membrane. We have isolated novel sec63 mutant alleles that display severe karyogamy defects. Disruption of the genes encoding other Sec63p-associated proteins (Sec71p and Sec72p) also results in karyogamy defects. A suppressor mutant (sos1-1) partially corrects the translocation defect but does not alleviate the karyogamy defect. sec61 and sec62 mutant alleles that cause similar or more severe protein translocation defects show no karyogamy defects. Taken together, these results suggest a direct role for Sec63p, Sec71p, and Sec72p in nuclear membrane fusion and argue against the alternative interpretation that the karyogamy defects result as an indirect consequence of the impaired membrane translocation of another component(s) required for the process. We propose that an ER/nuclear membrane protein complex composed of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear membrane fusion and requires ER luminally associated Kar2p for its function.     

Pmt-mediated O mannosylation stabilizes an essential component of the secretory apparatus, Sec20p, in Candida albicans

Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.     

Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites

Sac1 phosphoinositide (PI) phosphatases are essential regulators of PI-signaling networks. Yeast Sac1, an integral endoplasmic reticulum (ER) membrane protein, controls PI4P levels at the ER, Golgi, and plasma membrane (PM). Whether Sac1 can act in trans and turn over PI4P at the Golgi and PM from the ER remains a paradox. We find that Sac1-mediated PI4P metabolism requires the oxysterol-binding homology (Osh) proteins. The PH domain-containing family member, Osh3, localizes to PM/ER membrane contact sites dependent upon PM PI4P levels. We reconstitute Osh protein-stimulated Sac1 PI phosphatase activity in vitro. We also show that the ER membrane VAP proteins, Scs2/Scs22, control PM PI4P levels and Sac1 activity in vitro. We propose that Osh3 functions at ER/PM contact sites as both a sensor of PM PI4P and an activator of the ER Sac1 phosphatase. Our findings further suggest that the conserved Osh proteins control PI metabolism at additional membrane contact sites.     

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.     

Induction of lipid metabolic enzymes during the endoplasmic reticulum stress response in plants

The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays) floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.     

A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

The cortical endoplasmic reticulum (cER) of yeast underlies the plasma membrane (PM) at specific contact sites to enable a direct transfer of information and material between both organelles. During budding, directed movement of cER to the young bud followed by subsequent anchorage at its tip ensures the faithful inheritance of this organelle. The ER membrane protein Scs2p tethers the cER to the PM and to the bud tip through so far unknown receptors. We characterize Epo1p as a novel member of the polarisome that interacts with Scs2p exclusively at the cell tip during bud growth and show that Epo1p binds simultaneously to the Cdc42p guanosine triphosphatase–activating protein Bem3p. Deletion of EPO1 or deletion of BEM3 in a polarisome-deficient strain reduces the amount of cER at the tip. This analysis therefore identifies Epo1p as a novel and important component of the polarisome that promotes cER tethering at sites of polarized growth.     

Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae

Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.     

Phosphoinositide-binding domains: Functional units for temporal and spatial regulation of intracellular signalling

Inositol phospholipid (phosphoinositide) is a versatile lipid characterized by its isomer-specific localization, as well as its molecular diversity attributable to phosphorylation events. Phosphoinositides act as signal mediators in a spatially and temporally controlled manner. Information about the timing and location of their production is received by phosphoinositide-binding proteins and transmitted to multiple lines of intracellular events such as signal transduction, cytoskeletal rearrangement, and membrane trafficking. Among those proteins, a significant portion possess globular structural units, called domains, which are specialized for phosphoinositide binding. The pleckstrin homology (PH) domain was the first phosphoinositide-binding domain identified. It contains the largest number of members and is associated with the formation of signalling complexes on the plasma membrane. Recent studies identified other novel phosphoinositide-binding domains (Fab1p, YOTB, Vps27p, EEA1 (FYVE), Phox homology (PX), and epsin N-terminal homology (ENTH)), thus extending our knowledge of how the functional versatility of phosphoinositides is achieved.