Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum

Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at approximately 4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25 degrees C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity approximately 5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447-450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase.     

Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast

Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SEC61 (Deshaies, R. J., and R. Schekman. 1987. J. Cell Biol. 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.     

Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis

Usher syndrome (USH), the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER). Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.KEY WORDS: Harmonin, Cadherin23, Ift88, Myo7aa, Usher syndrome, Hair cell, Trafficking, ER stress, Zebrafish     

Mutant prion proteins are partially retained in the endoplasmic reticulum

Familial prion diseases are linked to point and insertional mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP to the infectious isoform. In this report, we have investigated the subcellular localization of PrP molecules carrying pathogenic mutations using immunofluorescence staining, immunogold labeling, and PrP-green fluorescent protein chimeras. To facilitate visualization of the mutant proteins, we have utilized a novel Sindbis viral replicon engineered to produce high protein levels without cytopathology. We demonstrate that several different pathogenic mutations have a common effect on the trafficking of PrP, impairing delivery of the molecules to the cell surface and causing a portion of them to accumulate in the endoplasmic reticulum. These observations suggest that protein quality control in the endoplasmic reticulum may play an important role in prion diseases, as it does in some other inherited human disorders. Our experiments also show that chimeric PrP molecules with the sequence of green fluorescent protein inserted adjacent to the glycolipidation site are post-translationally modified and localized normally, thus documenting the utility of these constructs in cell biological studies of PrP.     

Retrieval of transmembrane proteins to the endoplasmic reticulum

A COOH-terminal double lysine motif maintains type I transmembrane proteins in the ER. Proteins tagged with this motif, eg., CD8/E19 and CD4/E19, rapidly receive post-translational modifications characteristic of the intermediate compartment and partially colocalized to this organelle. These proteins also received modifications characteristic of the Golgi but much more slowly. Lectin staining localized these Golgi modified proteins to ER indicating that this motif is a retrieval signal. Differences in the subcellular distribution and rate of post-translational modification of CD8 maintained in the ER by sequences derived from a variety of ER resident proteins suggested that the efficiency of retrieval was dependent on the sequence context of the double lysine motif and that retrieval may be initiated from multiple positions along the exocytotic pathway.     

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Background  

Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.     

Protein transport across the endoplasmic reticulum membrane

A decisive step in the biosynthesis of many eukaryotic proteins is their partial or complete translocation across the endoplasmic reticulum membrane. A similar process occurs in prokaryotes, except that proteins are transported across or are integrated into the plasma membrane. In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex. Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid.     

Failure of leukotriene B4 to translocate calcium across phosphatidylcholine membranes

It has been reported that leukotriene B4 can translocate calcium across model membranes (Serhan et. al., (1982) J. Biol. Chem., 257: 4746). Such ionophoretic behavior could account for its biological effects. We have examined the effect of chromatographically pure leukotriene B4 on Ca2+ permeability when added exogenously at 3 microM to phosphatidylcholine liposomes and when incorporated at 5 mole % in the lipid mixture used to prepare liposomes. No effect was observed with either procedure. An oxidized preparation of leukotriene B4 stimulated calcium permeability, however, suggesting that oxidation may account for the previously reported ionophoretic behavior of leukotriene B4.     

An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.     

Genes required for completion of import of proteins into the endoplasmic reticulum in yeast

Yeast secretory mutants sec53 and sec59 define a posttranslational stage in the penetration of glycoprotein precursors into the endoplasmic reticulum (ER). In the previous report we showed that at the restrictive temperature (37 degrees C) these mutants accumulate enzymatically inactive and incompletely glycosylated forms of the secretory enzyme invertase and the vacuolar enzyme carboxypeptidase Y. Cell fractionation experiments reveal that these precursor forms remain firmly bound to the ER membrane. However, upon return to the permissive temperature (24 degrees C), the invertase precursors are glycosylated, become partially active, and are secreted. Thermoreversible conversion does not require protein synthesis, but does require energy. In contrast to the effect of these mutations, inhibition of oligosaccharide synthesis with tunicamycin at 37 degrees C causes irreversible accumulation of unglycosylated invertase. The effect of the drug is exaggerated by high temperature since unglycosylated invertase synthesized in the presence of tunicamycin at 25 degrees C is secreted. A portion of the invertase polypeptide accumulated at 37 degrees C is preserved when membranes from sec53 and sec59 are treated with trypsin. In the presence of Triton X-100 or saponin, the invertase is degraded completely. The protected fragment appears to represent a portion of the invertase polypeptide that is embedded in or firmly associated with the ER membrane. This association may develop early during the synthesis of invertase, so that in the absence of translocation, some of the completed polypeptide chain remains exposed on the cytoplasmic surface of the ER.     

Plant G proteins interact with endoplasmic reticulum luminal protein receptors to regulate endoplasmic reticulum retrieval

Maintaining endoplasmic reticulum (ER) homeostasis is essential for the production of biomolecules. ER retrieval, i.e., the retrograde transport of compounds from the Golgi to the ER, is one of the pathways that ensures ER homeostasis. However, the mechanisms underlying the regulation of ER retrieval in plants remain largely unknown. Plant ERD2‐like proteins (ERD2s) were recently suggested to function as ER luminal protein receptors that mediate ER retrieval. Here, we demonstrate that heterotrimeric G protein signaling is involved in ERD2‐mediated ER retrieval. We show that ERD2s interact with the heterotrimeric G protein Gα and Gγ subunits at the Golgi. Silencing of Gα, Gβ, or Gγ increased the retention of ER luminal proteins. Furthermore, overexpression of Gα, Gβ, or Gγ caused ER luminal proteins to escape from the ER, as did the co‐silencing of ERD2a and ERD2b. These results suggest that G proteins interact with ER luminal protein receptors to regulate ER retrieval.     

Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function

In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.     

Translocation of globin fusion proteins across the endoplasmic reticulum membrane in Xenopus laevis oocytes

We have studied the translocation of a normally cytoplasmic protein domain across the membrane of the endoplasmic reticulum in cell-free systems and in Xenopus laevis oocytes. Coding regions for the normally cytoplasmic protein globin were engineered in frame either 3' or 5' to the coding region for the signal sequence of either Escherichia coli b-lactamase or bovine preprolactin, respectively, in SP6 expression plasmids. RNA transcribed from these plasmids was microinjected into oocytes as well as translated in cell-free systems. We demonstrate that both in vivo and in vitro, a previously amino-terminal signal sequence can direct translocation of domains engineered to either side. Moreover, the domain preceding the signal sequence can be as large as that which follows it. While, in general, cell-free systems were found to faithfully reflect translocation events in vivo, our results suggest that a mechanism for clearance of signal peptides after cleavage is present in intact cells that is not reconstituted in cell-free systems.     

Chaperones of the endoplasmic reticulum are required for Ve1‐mediated resistance to Verticillium

The tomato receptor‐like protein (RLP) Ve1 mediates resistance to the vascular fungal pathogen Verticillium dahliae. To identify the proteins required for Ve1 function, we transiently expressed and immunopurified functional Ve1‐enhanced green fluorescent protein (eGFP) from Nicotiana benthamiana leaves, followed by mass spectrometry. This resulted in the identification of peptides originating from the endoplasmic reticulum (ER)‐resident chaperones HSP70 binding proteins (BiPs) and a lectin‐type calreticulin (CRT). Knock‐down of the different BiPs and CRTs in tomato resulted in compromised Ve1‐mediated resistance to V. dahliae in most cases, showing that these chaperones play an important role in Ve1 functionality. Recently, it has been shown that one particular CRT is required for the biogenesis of the RLP‐type Cladosporium fulvum resistance protein Cf‐4 of tomato, as silencing of CRT3a resulted in a reduced pool of complex glycosylated Cf‐4 protein. In contrast, knock‐down of the various CRTs in N. benthamiana or N. tabacum did not result in reduced accumulation of mature complex glycosylated Ve1 protein. Together, this study shows that the BiP and CRT ER chaperones differentially contribute to Cf‐4‐ and Ve1‐mediated immunity.     

Assembly of the endoplasmic reticulum phospholipid bilayer: the phosphatidylcholine transporter

Phospholipids are synthesized and concomitantly inserted on the cytoplasmic surface of the endoplasmic reticulum. Assembly of the phospholipid bilayer requires translocation to the lumenal monolayer. The hypothesis that rat liver microsomes contain a protein transporter, or "flippase," for phosphatidylcholine was tested by measuring the transport of sn-1,2,-dibutyroylphosphatidylcholine (diC4PC). This homolog retains the polar head group, the portion of the phospholipid unable to undergo spontaneous transmembrane movement in vesicles, and its water solubility permits application of standard transport methods. DiC4PC entered the lumenal compartment of microsomal vesicles. Transport was saturable and was dependent on time, amount of microsomes, and an intact permeability barrier. DiC4PC transport was inhibited by structural analogs (but not by sn-2,3-diC4PC) and by treatment of microsomes with proteases, N-ethylmaleimide, and trinitrobenzenesulfonic acid. These data suggest that the transmicrosomal movement of diC4PC is protein mediated. DiC4PC was not transported across PC vesicles or red cell membranes, where PC translocation is slow.     

Polypeptide translocation across the endoplasmic reticulum membrane.

Many polypeptides have been postulated to play direct roles in secretory protein translocation based on genetic criteria, cross-linking, and antibody inhibition. Much of the excitement in the next few years will come from the resolution of current controversies. What is the nature of the ribosome receptor, and is it essential for translocation? Is BiP required for translocation in mammalian cells? Are all of the polypeptides of signal peptidase and oligosaccharyltransferase required for catalytic function, or do some of them mediate steps of protein translocation? One of the best ways to resolve these problems will be to determine the importance of each in reconstituted translocation reactions by fractionation or immunodepletion, or by analysis in a purified reaction. Another approach is to identify homologues of these molecules in S. cerevisiae and to assess their importance in in vivo translocation. Several mechanistic questions remain to be addressed as well. Does the protein translocation apparatus consist of protein, or lipid, or both? How are integral membrane proteins inserted? How is the translocon gated to admit only unfolded or partially folded secretory polypeptides and to exclude cytoplasmic molecules? The answers to these questions will illuminate a basic enigma in cell biology that has remained unanswered for many years.     

ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.     

Oligomeric complexes involved in translocation of proteins across the membrane of the endoplasmic reticulum.

Proteins involved in protein translocation across the membrane of the endoplasmic reticulum assemble into different oligomeric complexes depending on their state of function. To analyse such membrane protein complexes we fractionated proteins of mammalian rough microsomes and analysed them using blue native PAGE and immunoblotting. Among the proteins characterised are the Sec61p complex, the oligosaccharyl transferase (OST) complex, the translocon-associated protein (TRAP) complex, the TRAM and RAMP4 proteins, the signal recognition particle (SRP) and the SRP receptor (SR). Interestingly, the RAMP4 protein, SR and OST complex display more than one oligomeric form.     

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.