Structure and assembly of the endoplasmic reticulum: biosynthesis and intracellular sorting of ERp61, ERp59, and ERp49, three protein components of murine endoplasmic reticulum

Rabbit antibodies have been prepared against ERp61, ERp59, and ERp49, three protein components of rough endoplasmic reticulum (RER) purified from mineral oil-induced plasmacytoma 315 (MOPC-315) tissue. Analysis of subcellular fractions of MOPC-315 tissue by an immunoprecipitation procedure demonstrated that all three endoplasmic reticulum proteins (ERps) were most enriched in the RER. Immunologically cross-reacting proteins of similar molecular weight have been detected in other eucaryotic cell lines. We have used these antibodies to study the post-translational processing and biosynthetic sorting of the three ERps in pulse-labeled MOPC-315 cells. No larger precursor forms of the ERps were detected and none of the ERps were found to possess asparagine-linked oligosaccharide moieties. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 cells to study the biosynthetic sorting of ERp61, ERp59 and ERp49 and have found no evidence to suggest that these proteins ever leave the endoplasmic reticulum. In addition, all three ERps appeared to have luminally exposed domains. ERp61 and ERp59 were entirely protected by the ER membrane in the absence of detergent, while ERp49 was a transmembrane protein that also possesses a cytoplasmically exposed domain. We have used the anti-ERp antibodies to quantitate the synthesis and accumulation of the three ERps during lipopolysaccharide (LPS)-induced lymphocyte differentiation. After 48 h of culture in the presence of LPS, the synthesis of ERp49 increased sixfold relative to that in control cells. The synthesis and membrane accumulation of ERp61 and ERp59 were less affected by the LPS treatment. Thus, membranes isolated from LPS-treated cells were enriched in ERp49 relative to those isolated from control cells.     

Structure and assembly of the endoplasmic reticulum. The synthesis of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes

Monospecific rabbit antibodies have been prepared against ERp72, ERp99, and ERp60, major protein components of a detergent-solubilized extract of endoplasmic reticulum purified from mineral oil-induced plasmacytoma 315 tissue. When subcellular fractions of mineral oil-induced plasmacytoma 315 tissue were assayed by an immunoprecipitation procedure, all three endoplasmic reticulum proteins (ERps) were found to be enriched in the rough endoplasmic reticulum. In murine lymphoid cells, the three ERps represent two major structural classes of protein. Both ERp72 and ERp60 contain no endoglycosidase H-sensitive, N-linked oligosaccharides. On the other hand, ERp99 is glycoprotein containing, in all likelihood, one endoglycosidase H-sensitive oligosaccharide. Immunologically cross-reacting proteins of similar molecular weight have also been detected in other eukaryotic cell lines. The anti-ERp antibodies were used to quantitate the synthesis and accumulation of the three ERps in splenic lymphocytes cultured in the presence and absence of bacterial lipopolysaccharide (Escherichia coli serotype B5:055) (LPS). In the presence of LPS, lymphocytes differentiate from resting cells into actively secreting cells. The synthesis of ERp72 and ERp99 increased 3- and 10-fold, respectively, in response to LPS. The synthesis of ERp60 does not change significantly. The turnover rates for these three proteins are similar in both control and LPS-treated lymphocytes. As a result, membranes isolated from LPS-treated cells are enriched in ERp72 and ERp99.     

Biosynthesis and processing of ribophorins in the endoplasmic reticulum

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.     

ERp99, an abundant, conserved glycoprotein of the endoplasmic reticulum, is homologous to the 90-kDa heat shock protein (hsp90) and the 94-kDa glucose regulated protein (GRP94)

We have isolated an expressible full-length cDNA clone encoding murine ERp99, an abundant, conserved transmembrane glycoprotein of the endoplasmic reticulum membrane. ERp99 is synthesized as a 92,475-kDa precursor containing 802 amino acids. It possesses a signal peptide of 21 amino acids which is cleaved cotranslationally. Analysis of the amino acid sequence deduced from the nucleotide sequence of the cDNA clone led us to propose a model for the orientation of ERp99 in the endoplasmic reticulum membrane. In this model, ERp99 possesses one membrane-spanning, stop transfer segment in the N-terminal region. The protein chain passes through the membrane only once, and approximately 75% of the protein remains on the cytoplasmic side of the ER membrane. Comparison of the ERp99 sequence to the sequence of other proteins revealed that ERp99 has extensive homology with the 90-kDa heat shock protein of Saccharomyces cerevisiae (hsp90) and the 83-kDa heat shock protein of Drosophila melanogaster. In addition, the N terminus of mature ERp99 is identical to that of the 94-kDa glucose regulated protein (GRP94) of mammalian cells.     

ERp72, an abundant luminal endoplasmic reticulum protein, contains three copies of the active site sequences of protein disulfide isomerase

We have cloned, sequenced, and expressed full length cDNA clones encoding two abundant, luminal endoplasmic reticulum proteins (ERp), ERp59/PDI and ERp72. ERp59/PDI has been identified as the microsomal enzyme protein disulfide isomerase (PDI). An analysis of the amino acid sequence of ERp72 showed that it shared sequence identity with ERp59/PDI at three discrete regions, having three copies of the sequences that are thought to be the CGHC-containing active sites of ERp59/PDI. Thus, ERp72 appears to be a newly described member of the family of CGHC-containing proteins. ERp59/PDI has the sequence KDEL at its COOH terminus while ERp72 has the related sequence KEEL. Removal of the KDEL of ERp59/PDI or the KEEL of ERp72 by in vitro mutagenesis techniques and subsequent analysis of the mutants in transient expression assays, showed that both sequences are endoplasmic reticulum retention signals for their respective proteins. The most dramatic difference in secretion between the wild type and the mutant forms of the protein was seen in the case of ERp72.     

Analysis of the retention signals of two resident luminal endoplasmic reticulum proteins by in vitro mutagenesis

Protein disulfide isomerase (PDI, ERp59), ERp72, and ERp61 are luminal proteins of the endoplasmic reticulum (ER) that are characterized by the presence of sequences corresponding to the active site regions of PDI. Each one of these proteins possesses a different COOH-terminal tetrapeptide ER retention signal. In order to investigate what other tetrapeptide sequences could serve as retention signals and to determine to what extent the function of the retention signal is modulated by the protein carrying the signal, we have constructed a set of mutants of two of these resident ER proteins, PDI and ERp72. In each of these proteins, the wild type tetrapeptide sequences were replaced by each member of the set of the 12 possible combinations consisting of (K,R,Q)-(D,E)-(D,E)-L. Analysis of the efficiency of retention of the variant proteins when each was transiently expressed in COS cells showed that the retention efficiencies vary with both the COOH-terminal sequence and with the protein that carries this sequence.     

Cell surface targeting of myelin oligodendrocyte glycoprotein (MOG) in the absence of endoplasmic reticulum molecular chaperones

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane glycoprotein that localizes to myelin sheaths in the central nervous system. MOG has important implications in multiple sclerosis, as pathogenic anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. As a membrane protein, MOG achieves its native structure in the endoplasmic reticulum where its folding is expected to be controlled by endoplasmic reticulum chaperones. Calnexin, calreticulin, and ERp57 are essential components of the endoplasmic reticulum quality control where they assist in the proper folding of newly synthesized glycoproteins. In this study, we show that expression of MOG is not affected by the absence of the endoplasmic reticulum quality control proteins calnexin, calreticulin, or ERp57. We also show that calnexin forms complexes with MOG and these interactions might be glycan-independent. Importantly, we show that cell surface targeting of MOG is not disrupted in the absence of the endoplasmic reticulum chaperones. This article is part of a special issue entitled: 11th European Symposium on Calcium.     

Separate roles and different routing of calnexin and ERp57 in endoplasmic reticulum quality control revealed by interactions with asialoglycoprotein receptor chains

The thiol oxidoreductase endoplasmic reticulum (ER)p57 interacts with newly synthesized glycoproteins through ternary complexes with the chaperones/lectins calnexin or calreticulin. On proteasomal inhibition calnexin and calreticulin concentrate in the pericentriolar endoplasmic reticulum-derived quality control compartment that we recently described. Surprisingly, ERp57 remained in an endoplasmic reticulum pattern. Using asialoglycoprotein receptor H2a and H2b as models, we determined in pulse-chase experiments that both glycoproteins initially bind to calnexin and ERp57. However, H2b, which will exit to the Golgi, dissociated from calnexin and remained bound for a longer period to ERp57, whereas the opposite was true for the endoplasmic reticulum-associated degradation substrate H2a that will go to the endoplasmic reticulum-derived quality control compartment. At 15 degrees C, ERp57 colocalized with H2b adjacent to an endoplasmic reticulum-Golgi intermediate compartment marker. Posttranslational inhibition of glucose excision prolonged association of H2a precursor to calnexin but not to ERp57. Preincubation with a low concentration (15 microg/ml) of the glucosidase inhibitor castanospermine prevented the association of H2a to ERp57 but not to calnexin. This low concentration of castanospermine accelerated the degradation of H2a, suggesting that ERp57 protects the glycoprotein from degradation and not calnexin. Our results suggest an early chaperone-mediated sorting event with calnexin being involved in the quality control retention of molecules bound for endoplasmic reticulum-associated degradation and ERp57 giving initial protection from degradation and later assisting the maturation of molecules that will exit to the Golgi.     

Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA

The mechanism by which endoplasmic reticulum (ER) stress proteins are induced by the accumulation of incompletely assembled or malfolded proteins in the ER is poorly understood. The 78-kDa glucose-regulated protein (BiP), one of the ER stress proteins, has often been detected in stable complexes with these accumulated proteins. We have transfected COS cells with an immunoglobulin (Ig) mu heavy chain expression plasmid. Expressed mu-chain accumulated in the cells and formed stable complexes with BiP. As a result, the synthesis of three ER stress proteins, BiP, the 94-kDa glucose-regulated protein (GRP94/ERp99), and ERp72, was increased as were their mRNA levels. In addition, the degradation rate of BiP was increased, possibly because of its interaction with mu-chain. Cotransfection of the mu-chain plasmid with an Ig lambda light chain expression plasmid resulted in the appearance of mu-chain in the media in a covalent complex with lambda-chain. An intracellular consequence of this was a reduction in the levels of BiP.mu-chain complex, and a diminished stimulation in the synthesis of the ER stress proteins. These results suggest that the BiP.mu-chain complex in the ER may be involved in the signaling pathway for the induction of ER stress proteins and may represent one regulatory mechanism operating in differentiating B-lymphocytes.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Recycling of proteins between the endoplasmic reticulum and Golgi complex.

Several lines of investigation have shown that protein transport from the endoplasmic reticulum to the Golgi is more complex than previously imagined. Dynamic sorting of both membrane and soluble proteins is now believed to occur on the cis side of the Golgi apparatus with some proteins returning to the endoplasmic reticulum while others travel onwards.     

COPII-dependent transport from the endoplasmic reticulum

The coat protein complex II (COPII) forms transport vesicles from the endoplasmic reticulum and segregates biosynthetic cargo from ER-resident proteins. Recent high-resolution structural studies on individual COPII subunits and on the polymerized coat reveal the molecular architecture of COPII vesicles. Other advances have shown that integral membrane accessory proteins act with the COPII coat to collect specific cargo molecules into ER-derived transport vesicles.     

ERp19 and ERp46, new members of the thioredoxin family of endoplasmic reticulum proteins

Using a proteomic analysis of the luminal environment of the endoplasmic reticulum (ER), we have identified 141 proteins, of which 6 were previously unknown. Two newly discovered ER luminal proteins, designated ERp19 and ERp46, are related to protein disulphide isomerase. Western and Northern blot analyses revealed that both ERp19 and ERp46 and their respective mRNAs are highly expressed in the liver as compared with other tissues. Both proteins were enriched in purified liver ER vesicles and were localized specifically to the ER in McA-RH7777 hepatocytes. Functional analysis with yeast complementation studies showed that ERp46 but not ERp19 can substitute for protein disulphide isomerase function in vivo.     

Protein transport from endoplasmic reticulum to the Golgi complex can occur during meiotic metaphase in Xenopus oocytes

We have previously shown that Xenopus oocytes arrested at second meiotic metaphase lost their characteristic multicisternal Golgi apparati and cannot secrete proteins into the surrounding medium. In this paper, we extend these studies to ask whether intracellular transport events affecting the movement of secretory proteins from the endoplasmic reticulum to the Golgi apparatus are also similarly inhibited in such oocytes. Using the acquisition of resistance to endoglycosidase H (endo H) as an assay for movement to the Golgi, we find that within 6 h, up to 66% of the influenza virus membrane protein, hemagglutinin (HA), synthesized from injected synthetic RNA, can move to the Golgi apparati in nonmatured oocytes; indeed after longer periods some correctly folded HA can be detected at the cell surface where it distributes in a nonpolarized fashion. In matured oocytes, up to 49% of the HA becomes endo H resistant in the same 6-h period. We conclude that movement from the endoplasmic reticulum to the Golgi can occur in matured oocytes despite the dramatic fragmentation of the Golgi apparati that we observe to occur on maturation. This observation of residual protein movement during meiotic metaphase contrasts with the situation at mitotic metabphase in cultured mammalian cells where all movement ceases, but resembles that in the budding yeast Saccharomyces cerevisiae where transport is unaffected.     

Drosophila arf72A acts as an essential regulator of endoplasmic reticulum quality control and suppresses autosomal-dominant retinopathy

The eukaryotic endoplasmic reticulum operates multiple quality control mechanisms to ensure that only properly folded proteins are exported to their final destinations via the secretory pathway and those that are not are destroyed via the degradation pathway. However, molecular mechanisms underlying such regulated exportation to these distinct routes are unknown. In this article, we report the role of Drosophila arf72A--the fly homologue of the mammalian Arl1 - in the quality checks of proteins and in the autosomal-dominant retinopathy. ARF72A localizes to the Golgi membranes of Drosophila photoreceptor cells, consistent with mammalian Arl1 localization in cell culture systems. A loss of arf72A function changes the membrane character of the endoplasmic reticulum and shifts the membrane balance between the endoplasmic reticulum and the Golgi complex toward the Golgi complex, resulting in over-proliferated Golgi complexes and accelerated protein secretion. Interestingly, our study indicated that more ARF72A localized on the endoplasmic reticulum in the ninaE(D1) photoreceptor cell, a Drosophila model of autosomal-dominant retinitis pigmentosa, compared to that in the wild-type. In addition, arf72A loss was shown to rescue the ninaE(D1)-related membrane accumulation and the rhodopsin maturation defect, and suppress ninaE(D1)-triggered retinal degeneration, indicating that rhodopsin accumulated in the endoplasmic reticulum bypasses the quality checks. While previous studies of ARF small GTPases have focused on their roles in vesicular budding and transport between the specific organelles, our findings establish an additional function of arf72A in the quality check machinery of the endoplasmic reticulum distinguishing the cargoes for secretion from those for degradation.     

Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein-protein and protein-lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail-anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER-Golgi boundary by simple receptor-independent mechanisms based on partitioning.     

Rer1, a putative transmembrane domain receptor responsible for targeting proteins to the endoplasmic reticulum

Summary Localization of resident proteins provides identity to subcellular compartments. Most proteins depend on a combination of both retention and retrieval to maintain their steady-state distribution. Rerl is a putative receptor protein mediating retrieval of membrane proteins of the endoplasmic reticulum. This retrieval relies on an unusual hydrophobic target sequence, the transmembrane domain. Apart from Rerl, coatomer is also required to retrieve escaped membrane proteins from the early Golgi region back to the endoplasmic reticulum. Current evidence suggests that the Rerl-mediated retrieval of membrane proteins is a general sorting pathway in eukaryotic cells contributing to the maintenance of compartmental identity in the early secretory pathway.     

Cotranslational cleavage of immunoglobulin light chain precursors by plasmacytoma microsomes.

Murine plasmacytoma endoplasmic reticulum which has been freed of ribosomes by EDTA treatment is capable of the cotranslational proteolytic processing of representative λ₁,λ₂, and k immunoglobulin light chain precursors. Messenger RNA fractions from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines were used to direct the synthesis of the light chain precursors in a cell-free system derived from Krebs II ascites cells. The precursor cleavage activity of the plasmacytoma membranes is comparable in activity and in characteristics to that of two well-defined membrane preparations: Krebs II ascites intracellular membranes (E. Szczesna and I. Boime, 1976, Proc. Nat. Acad. Sci. USA73, 1179–1183) and EDTA-treated rough endoplasmic reticulum from canine pancreas (34., 35., J. Cell Biol.67, 852–862). The efficiency of the cleavage reaction appears to be dependent upon the precursor being utilized as a substrate. An assay suitable for a preliminary characterization of the plasmacytoma membrane preparations is described.