GPI-anchor remodeling: potential functions of GPI-anchors in intracellular trafficking and membrane dynamics

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. GPI is synthesized and transferred to proteins in the endoplasmic reticulum. GPI-anchored proteins are then transported from the endoplasmic reticulum to the plasma membrane through the Golgi apparatus. GPI-anchor functions as a sorting signal for transport of GPI-anchored proteins in the secretory and endocytic pathways. After GPI attachment to proteins, the structure of the GPI-anchor is remodeled, which regulates the trafficking and localization of GPI-anchored proteins. Recently, genes required for GPI remodeling were identified in yeast and mammalian cells. Here, we describe the structural remodeling and function of GPI-anchors, and discuss how GPI-anchors regulate protein sorting, trafficking, and dynamics. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

The dual origin of Toxoplasma gondii N-glycans

N-Linked glycosylation is the most frequent modification of secreted proteins in eukaryotic cells that plays a crucial role in protein folding and trafficking. Mature N-glycans are sequentially processed in the endoplasmic reticulum and Golgi apparatus through a pathway highly conserved in most eukaryotic organisms. Here, we demonstrate that the obligate intracellular protozoan parasite Toxoplasma gondii independently transfers endogenous truncated as well as host-derived N-glycans onto its own proteins.Therefore, we propose that the apicomplexan parasite scavenges N-glycosylation intermediates from the host cells to compensate for the rapid evolution of its biosynthetic pathway, which is primarily devoted to modification of proteins with glycosylphosphatidylinositols rather than N-glycans.     

N-glycans and glycosylphosphatidylinositol-anchor act on polarized sorting of mouse PrP(C) in Madin-Darby canine kidney cells

The cellular prion protein (PrP(C)) plays a fundamental role in prion disease. PrP(C) is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrP(C) is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrP(C) and also replaced the GPI-anchor of PrP(C) by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrP(C) in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrP(C). Exchange of the PrP(C) GPI-anchor for the one of Thy-1 redirects PrP(C) to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrP(C), with the GPI-anchor being dominant over N-glycans.     

Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase

Translocation of proteins across membranes of the endoplasmic reticulum, mitochondrion, and chloroplast has been shown to be mediated by targeting signals present in the transported proteins. To test whether the transport of proteins into peroxisomes is also mediated by a peptide targeting signal, we have studied the firefly luciferase gene that encodes a protein transported to peroxisomes in both insect and mammalian cells. We have identified two regions of luciferase which are necessary for transport of this protein into peroxisomes. We demonstrate that one of these, region II, represents a peroxisomal targeting signal because it is both necessary and sufficient for directing cytosolic proteins to peroxisomes. The signal is no more than twelve amino acids long and is located at the extreme carboxy-terminus of luciferase. The location of the targeting signal for translocation across the peroxisomal membrane therefore differs from the predominantly amino-terminal location of signals responsible for transport across the membranes of the endoplasmic reticulum, chloroplast, or mitochondrion.     

HSP60 is transported through the secretory pathway of 3-MCA-induced fibrosarcoma tumour cells and undergoes N-glycosylation

There is increasing evidence localizes the mitochondrial chaperone heat shock protein (HSP)60, outside the cell, where it mediates interactions between immune cells and other body tissues. However, the mechanisms by which HSP60 is secreted into the extracellular environment are not fully understood. Recent studies have shown that HSP60 is actively released by a nonconventional secretion mechanism, the lipid raft-exosome pathway. In the present study, we show for the first time that HSP60, produced by 3-methylcholantrene-induced fibrosarcoma tumour cells, is secreted through the conventional endoplasmic reticulum-Golgi secretory pathway. Confocal microscopy using anti-TGN38 and anti-HSP60 antibodies together with monensin, a Golgi transport inhibitor, demonstrated the relocation of HSP60 to the Golgi of malignant cells but not primary fibroblast cells subjected to heat shock or fibroblast cell lines. Transmission electron microscopy, flow cytometry and cell fractionation of cell treated with brefeldin A, an inhibitor of endoplasmic reticulum to Golgi protein transport, further indicated that HSP60 is present both in the endoplasmic reticulum and the Golgi complex of malignant cells. We found a single mRNA with a mitochondrial targeting sequence encoding for HSP60 in the malignant cells but two HSP60 translation products, namely the native unmodified protein and a protein post-translationally modified by N-glycosylation. The N-glycans observed were composed of high-mannose structures and bi-, tri- and tetra-antennary complex type structures occupying sites of the three potential glycosylation sites present on HSP60. Accordingly, we propose that HSP60 in malignant cells is transported through the endoplasmic reticulum-Golgi secretion pathway, where it acquires N-glycans, and thus can affect the immunological properties of the proteins in the tumour microenvironment.     

Deficiency in ethanolamine plasmalogen leads to altered cholesterol transport

Plasmalogens are a major sub-class of ethanolamine and choline phospholipids in which the sn-1 position has a long chain fatty alcohol attached through a vinyl ether bond. These phospholipids are proposed to play a role in membrane fusion-mediated events. In this study, we investigated the role of the ethanolamine plasmalogen plasmenylethanolamine (PlsEtn) in intracellular cholesterol transport in Chinese hamster ovary cell mutants NRel-4 and NZel-1, which have single gene defects in PlsEtn biosynthesis. We found that PlsEtn was essential for specific cholesterol transport pathways, those from the cell surface or endocytic compartments to acyl-CoA/cholesterol acyltransferase in the endoplasmic reticulum. The movement of cholesterol from the endoplasmic reticulum or endocytic compartments to the cell surface was normal in PlsEtn-deficient cells. Also, vesicle trafficking was normal in PlsEtn-deficient cells, as measured by fluid phase endocytosis and exocytosis, as was the movement of newly-synthesized proteins to the cell surface. The mutant cholesterol transport phenotype was due to the lack of PlsEtn, since it was corrected when NRel-4 cells were transfected with a cDNA encoding the missing enzyme or supplied with a metabolic intermediate that enters the PlsEtn biosynthetic pathway downstream of the defect. Future work must determine the precise role that plasmalogens have on cholesterol transport to the endoplasmic reticulum.     

Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding

In this report we have extended our studies on a panel of vesicular stomatitis virus G proteins with altered glycosylation sites. These mutant proteins were generated by oligonucleotide-directed mutagenesis of the coding sequence to create new consensus sites for asparagine-linked oligosaccharide addition. We report that the intracellular transport of most of the mutant proteins is temperature-sensitive, implying a polypeptide folding step is affected. In addition, we find that the nonglycosylated G protein and those mutant proteins which lack oligosaccharides at the normal positions are subject to aberrant intermolecular disulfide bonding, leading to the accumulation of large complexes in the endoplasmic reticulum. These results imply that carbohydrate plays an indirect role in the intracellular transport of G protein.     

Proper protein folding in the endoplasmic reticulum is required for attachment of a glycosylphosphatidylinositol anchor in plants

Endoplasmic reticulum (ER) quality control processes recognize and eliminate misfolded proteins to maintain cellular protein homeostasis and prevent the accumulation of defective proteins in the secretory pathway. Glycosylphosphatidylinositol (GPI)-anchored proteins carry a glycolipid modification, which provides an efficient ER export signal and potentially prevents the entry into ER-associated degradation (ERAD), which is one of the major pathways for clearance of terminally misfolded proteins from the ER. Here, we analyzed the degradation routes of different misfolded glycoproteins carrying a C-terminal GPI-attachment signal peptide in Arabidopsis thaliana. We found that a fusion protein consisting of the misfolded extracellular domain from Arabidopsis STRUBBELIG and the GPI-anchor attachment sequence of COBRA1 was efficiently targeted to hydroxymethylglutaryl reductase degradation protein 1 complex-mediated ERAD without the detectable attachment of a GPI anchor. Non-native variants of the GPI-anchored lipid transfer protein 1 (LTPG1) that lack a severely misfolded domain, on the other hand, are modified with a GPI anchor and targeted to the vacuole for degradation. Impaired processing of the GPI-anchoring signal peptide by mutation of the cleavage site or in a GPI-transamidase-compromised mutant caused ER retention and routed the non-native LTPG1 to ERAD. Collectively, these results indicate that for severely misfolded proteins, ER quality control processes are dominant over ER export. For less severely misfolded proteins, the GPI anchor provides an efficient ER export signal resulting in transport to the vacuole.

Severely misfolded proteins carrying a glycosylphosphatidylinositol (GPI)-anchor attachment sequence undergo a stringent quality control process in the endoplasmic reticulum that prevents GPI anchoring.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded delta-opioid receptors at the cell surface

A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn(18) and Asn(33)) of the human delta-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn(33) was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the delta-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.     

Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface- expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double- immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.     

N-glycosylation at noncanonical Asn-X-Cys sequences in plant cells

The vesicular transport pathway in plant cells is often used for higher accumulation of recombinant proteins. In the endoplasmic reticulum, which acts as a gateway to the vesicular transport pathway, N-glycosylation occurs on specific Asn residues. This N-glycosylation in recombinant proteins must be carefully regulated as it can impact their enzymatic activity, half lives in serum when injected, structural stability, etc. In eukaryotic cells, including plant cells, N-glycans were found to be attached to Asn residues in Asn-X-Ser/Thr (X ≠ Pro) sequences. However, recently, N-glycosylations at noncanonical Asn-X-Cys sequences have been found in mammals and yeast. Our laboratory has discovered that N-glycans are attached to Asn residues at Asn-Thr-Cys sequences of double-repeated B subunit of Shiga toxin 2e produced in plant cells, the first reported case of N-glycosylation at a noncanonical Asn-X-Cys sequence in plant cells.     

Distinct but dispensable N-glycosylation of human CD69 proteins.

Human CD69 is uniquely glycosylated at typical (Asn-X-Ser/Thr) and atypical (Asn-X-Cys) motifs, which represents the molecular basis for the formation of CD69 homodimers and heterodimers. Here we examined the importance of N-glycosylation for the assembly and intracellular transport of CD69 proteins using mutant CD69 molecules that specifically lack typical and atypical N-glycan attachment motifs. These studies verify the importance of Cys residues in atypical triplet sequences for N-glycan addition to human CD69 proteins in the endoplasmic reticulum (ER). In addition, these data demonstrate that monoglycosylated CD69 proteins (bearing N-glycans exclusively at atypical or typical sites) and aglycosylated CD69 molecules (lacking N-glycans) efficiently dimerize in the ER and have similar stability as wild-type CD69 molecules. Finally, these results show that CD69 proteins lacking atypical or typical N-glycan addition sites are transported to the plasma membrane.     

Purification, biosynthesis and cellular localization of a major 125-kDa glycophosphatidylinositol-anchored membrane glycoprotein of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been shown to contain a major 125-kDa membrane glycoprotein which is anchored in the lipid bilayer by a glycophosphatidylinositol anchor. This protein was purified to near homogeneity and was used to raise a rabbit antibody. Biosynthesis of the 125-kDa protein was studied by immunoprecipitation of 35SO4-labeled material from wild-type cells or a secretion mutant (sec18) in which the vesicular traffic from the endoplasmic reticulum (ER) to the Golgi is blocked. The 125-kDa protein is first made in the ER as a 105-kDa precursor which already contains a glycophosphatidylinositol anchor and which is slowly transformed into the 125-kDa form upon chase (t1/2 approximately 10-15 min). The 105-kDa precursor can be reduced to an 83-kDa form by the enzymatic removal of N-glycans. The removal of N-glycans from the mature 125-kDa protein yields a 95-kDa species. Thus, removal of the N-glycans does not reduce the ER and mature forms to the same molecular mass, indicating that not only elongation of N-glycans but also another post-translational modification takes place during maturation. Selective tagging of surface proteins by treatment of 35SO4-labeled cells with trinitrobenzene sulfonic acid at 0 C followed by immunoprecipitation of the tagged proteins shows that the 125-kDa protein, but not the 105-kDa precursor, becomes transported to the cell surface. This tagging of cells after various lengths of chase also shows that the surface appearance of the protein is biphasic with about one half of the mature 125-kDa protein remaining intracellular for over 2 h. Glycosylation and/or glycophosphatidylinositol anchor addition is important for the stability of the 125-kDa protein since the protein remains undetectable in sec53, a temperature-sensitive mutant which does not make GDP-mannose at 37 C and does not add glycophosphatidylinositol anchors at 37 degrees C.     

The arrangement of the Golgi complex beads is not controlled by the cytoskeleton

Exposure of insect fat body to treatments which disrupt microtubules (colchicine, vinblastine sulfate and cold treatment) blocks intracellular transport between the Golgi complex and the plasma membrane but does not affect Golgi complex bead rings or transport from rough endoplasmic reticulum to the Golgi complex. Drugs which disrupt microfilaments (cytochalasins B and D) do not affect the bead rings or intracellular transport of secretory proteins at any level. Thus, intracellular transport between the rough endoplasmic reticulum and the Golgi complex and the arrangement of the beads in rings are both independent of the cytoskeleton. The ring arrangement is presumably maintained by interconnection(s) with rough endoplasmic reticulum membrane.     

Post-translational glycosylation of coronavirus glycoprotein E1: inhibition by monensin.

The intracellular sites of biosynthesis of the structural proteins of murine hepatitis virus A59 have been analyzed using cell fractionation techniques. The nucleocapsid protein N is synthesized on free polysomes, whereas the envelope glycoproteins E1 and E2 are translated on the rough endoplasmic reticulum (RER). Glycoprotein E2 present in the RER contains N-glycosidically linked oligosaccharides of the mannose-rich type, supporting the concept that glycosylation of this protein is initiated at the co-translational level. In contrast, O-glycosylation of E1 occurs after transfer of the protein to smooth intracellular membranes. Monensin does not interfere with virus budding from the membranes of the endoplasmic reticulum, but it inhibits virus release and fusion of infected cells. The oligosaccharide side chains of E2 obtained under these conditions are resistant to endoglycosidase H and lack fucose suggesting that transport of this glycoprotein is inhibited between the trans Golgi cisternae and the cell surface. Glycoprotein E1 synthesized in the presence of monensin is completely carbohydrate-free. This observation suggests that the intracellular transport of this glycoprotein is also blocked by monensin.     

Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.     

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.     

The intracellular fate of a recombinant protein is tissue dependent

Recombinant proteins directed to the secretory pathway in plants require a signal peptide for entry into the endoplasmic reticulum. In the absence of further targeting information, such proteins are generally secreted via the default pathway to the apoplast. This has been well documented in protoplasts and leaf tissue, but the trafficking of recombinant proteins in seeds and other storage tissues has rarely been investigated. We used Aspergillus niger phytase as a model glycoprotein to compare the intracellular fate of a recombinant protein in the leaves and seeds of rice (Oryza sativa). Using fluorescence and electron microscopy we showed that the recombinant protein was efficiently secreted from leaf cells as expected. In contrast, within endosperm cells it was retained in endoplasmic reticulum-derived prolamin bodies and protein storage vacuoles. Consistent with our immunolocalization data, the phytase produced in endosperm cells possessed oligomannose and vacuolar-type N-glycans [Man(3)(Xyl)(Fuc)GlcNAc(2)], whereas the phytase produced in leaves contained predominantly secretion-type N-glycans [GlcNAc(2)Man(3)(Xyl)(Fuc)GlcNAc(2)]. The latter could not be detected in preparations of the endosperm-derived phytase. Our results show that the intracellular deposition and modification of a recombinant protein is tissue dependent.     

Carboxy-terminal SEKDEL sequences retard but do not retain two secretory proteins in the endoplasmic reticulum

The sequence Ser-Glu-Lys-Asp-Glu-Leu (SEKDEL) has been shown to be a signal which leads to retention of at least two proteins in the endoplasmic reticulum of animal cells (Munro and Pelham, 1987). In this study we tested the function of this signal by appending it to two secretory proteins, rat growth hormone and the alpha subunit of human chorionic gonadotrophin (hCG-alpha). We used oligonucleotide-directed mutagenesis and expression to generate proteins with SEKDEL added to the exact COOH termini and then carried out a detailed analysis of their transport in monkey COS cells. We found that transport was not blocked for either protein, but rather that the half-time for secretion was increased about sixfold for both proteins. Analysis of oligosaccharide processing on hCG-alpha-SEKDEL and indirect immunofluorescence microscopy on cells expressing both proteins was consistent with a retardation of transport between the endoplasmic reticulum and the Golgi apparatus. A change in the last amino acid of the SEKDEL sequence from Leu to Val abolished the retardation almost completely, suggesting a highly specific interaction of the sequence with a receptor. A change in the first amino acid had little or no effect on retardation. We conclude that the SEKDEL signal can have strong effects on reducing the rate of protein exit from the endoplasmic reticulum without generating absolute retention. Presumably other features of protein structure must be important to generate absolute retention.