C-terminal Endoplasmic Reticulum Retention Signal Research Articles

Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids (PUFA) are key to the biosynthesis of the long-chain polyunsaturated fatty acids (LC-PUFA). Here we report on the molecular cloning, tissue distribution, functional characterization and nutritional regulation of a elovl5 gene from Nibea coibor. The full-length cDNA was 1315 bp, including a 5-untranslated region (UTR) of 134 bp, a 3-UTR of 296 bp and an open reading frame of 885 bp, which specified a peptide of 294 amino acids. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of microsomal fatty acyl elongases, including the so-called histidine box (HXXHH), the canonical C-terminal endoplasmic reticulum retention signal, several predicted transmembrane regions and other highly conserved motifs. Expression of elovl5 was strongly observed in stomach, and more weakly in kidney, spleen, intestine, brain, eye, liver, gill, muscle and heart. Functional characterization revealed that the chu's croaker Elovl5 was able to elongate both C18 and C20 PUFA substrates. Nutritional study indicated that the hepatic expression of elovl5 could be up-regulated by low dietary n−3 LC-PUFA. These results may contribute to better understanding the LC-PUFA biosynthetic pathway and regulation mechanism in chu's croaker.

Characterization of the Structural and Functional Determinants of MANF/CDNF in Drosophila In Vivo Model

Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo.

Channel-Opening Kinetic Mechanism of Wild-Type GluK1 Kainate Receptors and a C-Terminal Mutant

GluK1 is a kainate receptor subunit in the ionotropic glutamate receptor family and can form functional channels when expressed, for instance, in HEK-293 cells. However, the channel-opening mechanism of GluK1 is poorly understood. One major challenge to studying the GluK1 channel is its apparent low level of surface expression, which results in a low whole-cell current response even to a saturating concentration of agonist. A low level of surface expression is thought to be contributed by an endoplasmic reticulum (ER) retention signal sequence. When this sequence motif is present as in the C-terminus of wild-type GluK1-2b, the receptor is significantly retained in the ER. Conversely, when this sequence is either lacking, as in wild-type GluK1-2a (i.e., a different alternatively spliced isoform at the C-terminus), or disrupted, as in a GluK1-2b mutant (i.e., R896A, R897A, R900A, and K901A), there is a higher level of surface expression and a greater whole-cell current response. Here we characterize the channel-opening kinetic mechanism for these three GluK1 receptors expressed in HEK-293 cells by using a laser-pulse photolysis technique. Our results show that wild-type GluK1-2a, wild-type GluK1-2b, and the GluK1-2b mutant have identical channel opening and channel closing rate constants. These results indicate that the amino acid sequence near or within the C-terminal ER retention signal sequence, which affects receptor trafficking and/or expression, does not affect channel gating properties. Furthermore, as compared with the GluK2 kainate receptor, the GluK1 channel is faster to open, close, and desensitize by at least 2-fold, yet the EC(50) value of GluK1 is similar to that of GluK2.

Isolation and Characterization of a Temperature-Regulated Microsomal Oleate Desaturase Gene (CtFAD2-1) from Safflower (Carthamus tinctorius L.)

A cDNA sequence, designated CtFAD2-1 and putatively encoding a delta-12 fatty acid desaturase, was isolated from total RNA of immature normal-type safflower (Carthamus tinctorius L.) seeds using a PCR approach. The deduced amino acid sequence showed three histidine boxes characteristic of all membrane-bound desaturase and a C-terminal endoplasmic reticulum retention signal. Phylogenetic analysis indicated that CtFAD2-1 is grouped with other plant FAD2 sequences which are specifically or highly expressed in developing seeds. Functional expression of the corresponding CtFAD2-1 cDNA in yeast confirmed that it encodes a microsomal oleate desaturase, partially converting oleic fatty acid to linoleic fatty acid. Our transcriptional analysis suggested that CtFAD2-1 is constitutively expressed, with maximum transcript accumulation, in developing seeds. The sequence of this microsomal oleate desaturase gene was also obtained from high oleic safflower genotypes, CtFAD2-1′. The comparison between the coding regions of CtFAD2-1 and CtFAD2-1′ revealed that a deletion of cytosine (C) exists at the position +603 bp of CtFAD2-1′. Analysis of transcript level showed that the expression of CtFAD2-1′ in high oleic acid genotype is significantly lower than that in normal genotypes during seed development. These results suggested that the change of CtFAD2-1′ gene sequence results in the deactivation and lower transcription of delta-12 fatty acid desaturase in high oleic safflower genotypes.

Molecular Cloning and Expression Analysis of Genes Encoding Two Microsomal Oleate Desaturases (FAD2) from Safflower (Carthamus tinctorius L.)

The endoplasmic reticulum (ER)-associated oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. In safflower, a seed-type FAD2 gene (CtFAD2-1) has been isolated previously. In this study, two different cDNA sequences, designated CtFAD2-2 and CtFAD2-3, encoding two microsomal oleate desaturases have been isolated from safflower (Carthamus tinctorius L.) using a polymerase chain reaction (PCR) approach. Both deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and possess a C-terminal ER retention signal. Phylogentic analysis shows that CtFAD2-2 is grouped with other house-keeping type FAD2 sequences from oil crops, while CtFAD2-3 does not belong to seed type or house-keeping type FAD2. Real-time PCR analysis showed that CtFAD2-2 gene is constitutively expressed in both vegetative tissues and developing seeds, with higher transcript levels in roots, stems and petioles. Transcript of CtFAD2-3 was strongly detected in developing seeds, showing low levels in vegetative tissues. In response to low-temperature stress, CtFAD2-2 and CtFAD2-3 in different tissues showed a different behavior (up- or down-regulated). The transcript of CtFAD2-2 was dramatically decreased in roots, stems and petioles under low temperature, while CtFAD2-3 showed a significant increase in these tissues. On the contrary, in leaves, the strongly enhanced expression of CtFAD2-2 was observed under low temperature, while the transcript of CtFAD2-3 showed a little decrease.

Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.

The Ile128Thr polymorphism influences stability and ligand binding properties of the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTTP) is essential for the assembly of VLDLs. We recently observed that a polymorphism in the MTTP promoter (-493G>T), which is in allelic association with an isoleucine-to-theronine substitution at position 128 (Ile128Thr) in the expressed protein, confers an increased risk of coronary heart disease. Two variant proteins comprising amino acids 16-297 of intact MTTP, MTTP(N)-Ile128 and MTTP(N)-Thr128, had similar native secondary structure content, as judged by circular dichroism. However, the thermal stability of MTTP(N)-Thr128 was greatly reduced, and this protein was also more extensively cleaved in limited proteolysis experiments compared with MTTP(N)-Ile128; both of these findings support a less compact fold. On adding LDL, which includes natively folded apolipoprotein B (apoB), decreased stability of the MTTP(N)-Thr128-LDL complex was observed compared with that of the MTTP(N)-Ile128-LDL complex. In a refined model of the N-terminal domain of MTTP, residue 128 is located in a surface-exposed position, in the same region as an identified MTTP binding site in the homologous apoB protein. Thus, the Ile128Thr polymorphism confers reduced structural stability, leading to decreased binding of MTTP to LDL particles. Because the major MTTP binding target on LDL is apoB, the Ile128Thr polymorphism could target the MTTP-apoB interaction.

Molecular cloning and characterization of genes encoding two microsomal oleate desaturases ( FAD2) from olive

Two different cDNA sequences, designated OepFAD2-1 and OepFAD2-2, encoding two microsomal oleate desaturases (FAD2) have been isolated from olive ( Olea europaea cv. Picual) using a PCR approach. Both deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and possess a C-terminal endoplasmic reticulum retention signal. Phylogenetic analysis shows that OepFAD2-1 and OepFAD2-2 are grouped with other plant FAD2 sequences. Functional expression of the corresponding FAD2 cDNAs in yeast confirmed that they encode microsomal oleate desaturases. Genomic Southern blot analysis is consistent with the presence of at least two copies of each OepFAD2 gene in the olive genome. OepFAD2-1 transcript was strongly detected in very young seeds and in leaves, showing low levels in mesocarps, while the transcript of the OepFAD2-2 gene was moderately expressed in developing seeds, ripening mesocarp and leaves. These expression data suggest differential functions for the two olive microsomal oleate desaturase genes, with FAD2-1 possibly responsible for the desaturation of reserve lipids in the young seed, while FAD2-2 may be mainly involved in storage lipid desaturation in the mature seeds and the mesocarp.

Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in Leishmania mexicana

Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.

Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Trafficking of wheat gluten proteins in transgenic tobacco plants: gamma-gliadin does not contain an endoplasmic reticulum-retention signal.

Wild-type and mutated forms of the wheat (Triticum aestivum L.) storage protein gamma-gliadin were expressed in transgenic tobacco (Nicotiana tabacum L. cv. NVS) under the control of the 35S cauliflower mosaic virus (CaMV) promoter in order to determine what, if any, endogenous targeting signals are present in the wild-type gamma-gliadin protein. The mutant forms of the protein were modified by the addition of a KDEL or HDEL C-terminal endoplasmic reticulum-retention signal, or the addition of a C-terminal propeptide from barley lectin which has been shown to be necessary and sufficient for targeting to the vacuole. Only modified forms of the protein accumulated in leaves of transgenic tobacco, although the transcript levels were similar for all the constructs. Pulse-chase analysis indicated that whereas the wild-type gamma-gliadin was rapidly turned over in tobacco leaves, KDEL and HDEL forms were highly stable. The vacuolar-signal mutant protein accumulated in tobacco leaves, but migrated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a lower mobility than wild-type gamma-gliadin, due in part to glycosylation of the C-terminal propeptide. The vacuolar-signal mutant protein was turned over slowly in tobacco, perhaps indicating a poor level of transport competence. When pulse-chase analysis was carried out on protoplasts isolated from tobacco plants expressing wild-type gamma-gliadin, but in the presence of Brefeldin A, gamma-gliadin was seen to accumulate. Taken together, these results indicate that gamma-gliadin is targeted to the vacuole in transgenic tobacco plants and does not contain any structural determinants which confer retention in the endoplasmic reticulum.

Seed-specific immunomodulation of abscisic acid activity induces a developmental switch.

A single-chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a 'wilty' phenotype, was put under control of the seed-specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild-type plants apart from their seeds. Anti-ABA scFv embryo development differed markedly from wild-type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild-type embryos. Anti-ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti-ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild-type seeds but calculated free ABA levels were near-zero until 21 days after pollination. We show for the first time seed-specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.

DNAs encoding ricin toxin A chain (RTA), with or without a C-terminal endoplasmic reticulum retention signal KDEL, were subcloned into pGEX2T bacterial expression plasmid. After transformation of JM105 E. coli cells and induction with isopropylthio-beta-galactoside (IPTG), fusion proteins were bound to an immobilized glutathione matrix and recombinant ricin A chains released with thrombin. Both recombinant wild-type RTA and RTA with KDEL had immunological reactivity and catalytic activity indistinguishable from plant RTA. The bacterial RTA products reassociated with plant ricin B chain (RTB) similarly to plant RTA. Cell cytotoxicities were measured on seven cell lines for each A-chain and heterodimer. Although KDEL sequences enhanced cytotoxicity in most cases, significant variability was observed. In each case, addition of KDEL enhanced A-chain cytotoxicity more than holotoxin cytotoxicity. Three cell lines showed reduced KDEL enhancement of both RTA and ricin cytotoxicity. The concentration of KDEL receptor was examined on each cell line by immunofluorescence microscopy with an antireceptor monoclonal antibody. Differences in sensitivity to KDEL-containing toxins correlated with altered distribution of KDEL receptor between endoplasmic reticulum (ER) and Golgi compartments.

Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.

The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.

A pathogen-induced gene of barley encodes a HSP90 homologue showing striking similarity to vertebrate forms resident in the endoplasmic reticulum.

The full-length nucleotide sequence of a barley (Hordeum vulgare L.) leaf mRNA, found to increase rapidly in amount during infection attempts by the powdery mildew fungus (Erysiphe graminis DC. ex Mérat), is reported. The mRNA encodes a polypeptide of 809 amino acid residues which, by sequence comparison, was identified as a member of the 90 kDa heat shock protein (HSP90) family. The encoded protein most resembles the endoplasmic reticulum (ER) resident HSP90 protein, the 94 kDa glucose-regulated protein (GRP94) of vertebrates, as it possesses both the characteristic N-terminal domain including a signal peptide sequence and the C-terminal ER retention signal (Lys-Asp-Glu-Leu). A transcript cross-hybridizing at high stringency accumulated rapidly in leaves upon heat shock treatment. Genomic DNA blot analysis indicated the presence of a family of related genes in the barley genome.

Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide-isomerase and a phosphatidylinositol-specific phospholipase C.

cDNA clones containing sequence similarity to the multifunctional vertebrate protein disulfide-isomerase (PDI, EC 5.3.4.1) were isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA sequence encoding human PDI. The polypeptide encoded by a clone designated B2 consisted of 512 amino acids and was characterized by a 24-amino acid hydrophobic leader sequence, two regions with absolute identity to the vertebrate PDI active site (Ala-Pro-Trp-Cys-Gly-His-Cys-Lys), and a C-terminal endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu). The overall identity of the B2 sequence to that of human PDI was 35% at the amino acid level (79% when conservative substitutions were included) and 39% at the nucleotide level; this included homology between B2 and the region of human PDI believed to be involved in binding estrogens. The deduced amino acid sequence of B2 was also 35% identical to that of a rat form I phosphatidylinositol-specific phospholipase C. Lysates from Escherichia coli cells harboring an expression plasmid bearing the B2 sequence contained significantly elevated levels of PDI activity. Southern analysis indicated the presence of a small PDI-related gene family in alfalfa, of which B2 appeared to correspond to a single gene. An approximately 2-kilobase B2 transcript was expressed in all alfalfa organs tested. In alfalfa cell suspension cultures, B2 transcripts were strongly induced by tunicamycin but not by exposure to fungal elicitor.

Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol

To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.