The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.     

The endoplasmic reticulum and the unfolded protein response

The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.     

Membrane protein integration into the endoplasmic reticulum

Most integral membrane proteins are targeted, inserted and assembled in the endoplasmic reticulum membrane. The sequential and potentially overlapping events necessary for membrane protein integration take place at sites termed translocons, which comprise a specific set of membrane proteins acting in concert with ribosomes and, probably, molecular chaperones to ensure the success of the whole process. In this minireview, we summarize our current understanding of helical membrane protein integration at the endoplasmic reticulum, and highlight specific characteristics that affect the biogenesis of multispanning membrane proteins.     

Regulation of immature protein dynamics in the endoplasmic reticulum

The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins. Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates. However, it is unclear how immature protein dynamics are regulated. In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase. The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy. The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP. Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature. This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion. Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching. In contrast, terminally misfolded tyrosinase synthesized in cells in which alpha-glucosidases were inhibited showed extensive immobilization. Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.     

Glucosidase II, a protein of the endoplasmic reticulum with high mannose oligosaccharide chains and a rapid turnover

Glucosidase II is regarded as a resident protein of the endoplasmatic reticulum. The enzyme removes alpha-1-3-linked glucose from high mannose oligosaccharides N-linked to asparagine residues of glycoproteins. Monospecific antibodies raised against the pig kidney enzyme are used to study the metabolism of the enzyme in a rat hepatoma cell line. These antiglucosidase II antibodies specifically immune precipitate glucosidase II as a 100,000-Da species from [35S]methionine-labeled cells. In addition, protein blotting and immune staining of cell extracts from both rat liver and human and rat hepatoma cell lines show identity in apparent Mr (100,000). Glucosidase II synthesized in the presence of tunicamycin is approximately 94,000 Da, indicating the presence of one or more N-linked oligosaccharide chains. Cell-free protein synthesis of rat hepatoma total RNA demonstrates that glucosidase II is synthesized as a slightly higher molecular weight species as compared to the polypeptide synthesized in whole cells in the presence of tunicamycin, indicating that the enzyme has a cleavable signal sequence. Using a pulse-chase protocol, the apparent molecular weight does not change upon longer chase periods. In addition, the 100,000-Da protein remains sensitive to endo-beta-N-acetylglucosaminidase H regardless of prolonged chase periods. The cells incorporate [3H]mannose into the enzyme; after release with endo-beta-N-acetylglucosaminidase H, most of the radioactivity comigrates with Glc1-Man9-GlcNAc on a gel filtration column. Phase separation in Triton X-114 shows a partition between the aqueous and the Triton phase, the major portion being separated in the aqueous phase. In rat hepatoma cells glucosidase II has a half-life of 50 min. This value is not altered if the cells are grown in the presence of monensin nor of methyl-deoxynoijirimycin. However, tunicamycin and low concentrations or primaquine (raising the pH of acidic compartments) causes a 100% increase in half-life of glucosidase II. We conclude that glucosidase II is a hydrophilic, probably not a transmembrane membrane, protein with a short half-life. It is the first example of an oligosaccharide-processing enzyme not being an integral membrane protein.     

The protein translocation machinery of the endoplasmic reticulum

The rough endoplasmic reticulum (r.e.r.) has been postulated to possess a single translation-coupled translocation system (in multiple copies) that effects signal sequence-mediated translocation of all secretory and lysosomal proteins and integration of all integral membrane proteins whose port of entry is the rough endoplasmic reticulum (G. Blobel 1980 Proc. natn. Acad. Sci. U.S.A. 77, 1496-1500). Two proteins have been isolated that are components of the r.e.r. translocation system. Their properties and function in protein translocation across and integration into membranes are discussed.     

Versatility of the endoplasmic reticulum protein folding factory

The endoplasmic reticulum (ER) is dedicated to import, folding and assembly of all proteins that travel along or reside in the secretory pathway of eukaryotic cells. Folding in the ER is special. For instance, newly synthesized proteins are N-glycosylated and by default form disulfide bonds in the ER, but not elsewhere in the cell. In this review, we discuss which features distinguish the ER as an efficient folding factory, how the ER monitors its output and how it disposes of folding failures.     

Signals and mechanisms for protein retention in the endoplasmic reticulum

After their co-translational insertion into the ER lumen or the ERmembrane, most proteins are transported via the Golgi apparatus downstreamon the secretory pathway while a few protein species are retained in theER. Polypeptide retention in the ER is either signal-independent or dependson specific retention signals encoded by the primary sequence of thepolypeptide. A first category, i.e. the newly synthesized polypeptides thatare unable to reach their final conformation, are retained in the ER wherethis quality control generally results in their degradation. A secondcategory, namely the ER-resident proteins escape the bulk flow of secretiondue to the presence of a specific N- or C-terminal signal that interactswith integral membrane or soluble receptors. ER retention of solubleproteins mediated by either KDEL, HDEL or related sequences and membranereceptors has been relatively well characterized in plants. Recent effortshas been relatively well characterized in plants. Recent efforts have aimedat a characterization of the retention signal(s) of type I membraneproteins in the plant ER.     

Tail-anchored membrane protein insertion into the endoplasmic reticulum

Membrane proteins are inserted into the endoplasmic reticulum (ER) by two highly conserved parallel pathways. The well-studied co-translational pathway uses signal recognition particle (SRP) and its receptor for targeting and the SEC61 translocon for membrane integration. A recently discovered post-translational pathway uses an entirely different set of factors involving transmembrane domain (TMD)-selective cytosolic chaperones and an accompanying receptor at the ER. Elucidation of the structural and mechanistic basis of this post-translational membrane protein insertion pathway highlights general principles shared between the two pathways and key distinctions unique to each.     

Endoplasmic-reticulum-associated protein degradation inside and outside of the endoplasmic reticulum

Summary Newly synthesized polypeptides that enter the endomembrane system encounter a folding environment in the lumen of the endoplasmic reticulum (ER) constituted by enzymes, lectinlike proteins, and molecular chaperones. The folding process is under scrutiny of this abundant catalytic machinery, and failure of the new arrivals to assume a stable and functional conformation is met with targeting to proteolytic destruction, a process which has been termed ER-associated degradation (ERAD). In recent years it became clear that, in most cases, proteolysis appears to take place in the cytosol after retro-translocation of the substrate proteins from the ER, and to depend on the ubiquitin-proteasome pathway. On the other hand, proteolytic activities within the ER that have been widely neglected so far may also contribute to the turnover of proteins delivered to ERAD. Thus, ERAD is being deciphered as a complex process that requires communication-dependent regulated proteolytic activities within both the ER lumen and the cytosol. Here we discuss some recent findings on ERAD and their implications on possible mechanisms involved.Abbreviations lAT alpha-1-antitrypsin - apoB apolipoprotein B - BiP immunoglobulin-heavy-chain-binding protein - CFTR cystic fibrosis transmembrane conductance regulator - CPY carboxypeptidase Y - ER endoplasmic reticulum - ERAD ER associated degradation - HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A - MHC major histocompatibility complex - PDI protein disulflde isomerase - TCR T cell antigen receptor     

Control of protein topology at the endoplasmic reticulum.

I have described recent work that supports several conclusions that might not have been previously expected: first, that stop transfer, like the initiation of translocation, is receptor-mediated; second, that at least some of the topology-determining events at the ER membrane can be regulated (an example is provided where regulation may occur developmentally [PrP] and a possible example where receptor interactions for stop transfer seem to have been dissociated from those of integration in the membrane, in the course of evolution [apo B]); third, that these variations on the universal mechanism of eukaryotic secretory and transmembrane protein biogenesis can occur either through the variations in sequences presented to the common machinery of translocation or through variations in the machinery with which these sequences interact. Thus, on the one hand, at least some of these variations are directed by signal and stop transfer sequence subtypes and, on the other hand, in at least one case, a special cytoplasmic factor distinct from the core machinery for chain translocation also seems to be involved (RRL cytosolic factor effect on PrP topology) in the special handling of the STE stop transfer sequence subtype. In another case, the conserved universal machinery is engaged by a protein (apo B) to carry out an unusual, if not unique, mechanism presumably related to the lipid carrying role of this soluble secretory protein. Whether stop transfer sequence subtypes are involved here remains to be demonstrated, but it is a tempting hypothesis. Taken together, these findings suggest that the ER is more than a barrier to be overcome in protein export. In some cases, it plays a regulatory role in gene expression (e.g., alternate fates of PrP), and in other cases, it plays a role as a specialized assembly line for biogenesis of proteins with unusual properties. It seems likely that many other examples of proteins using these two mechanisms will be found, as well as entirely different variations on the mechanisms of protein biogenesis. A common conceptual theme is likely to be that they are all directed by discrete sequences within the particular newly synthesized proteins engaging both/either the common and/or distinctive component of the cellular machinery for protein biogenesis.     

Phosphatidylethanolamine in liver mitochondria and endoplasmic reticulum: molecular species distribution and turnover.

Phosphatidylethanolamine from mitochondria and microsomes of guinea pig liver was separated by thin-layer chromatography into eight different classes differing in degree of unsaturation. The fatty acid compositions and molar proportions of each class isolated from microsomes were very similar to the corresponding class in mitochondria. In both organelles about half of the total was dienoic species while tetraenes comprised approximately 20%. Stearic acid was the major saturated fatty acid and in each membrane a greater selectivity for stearate over palmitate was found in each sub-class of phosphatidylcholine. Following the intraperitoneal injection of [2 minus-3 H]glycerol, the labelling of each molecular class of phosphatidylethanolamine showed very similar progressions in microsomes and mitochondria over a 3 h interval. In both organelles the highest relative specific activity was attained by penta-plus hexaenoic classes, while the large dienoic class had the lowest relative activity, which, however, increased with time. Analysis of the dienoic class of phosphatidyl-ethanolamine from whole liver showed it to be constituted by a rapidly turning over palmitoyl-linoleoyl fraction and a slowly labelled stearoyl-linoleoyl fraction, a pattern also exhibited by dienoic phosphatidylcholines. The similarities in profile of molecular classes of phosphatidylethanolamine and in the kinetics of labelling in vivo point to a close metabolic relation between the lipids of both organelles, suggestive of a transfer of different molecular classes at comparable rates from the endoplasmic reticulum, the site of synthesis, to the mitochondria. This is consistent with numerous other studies in vitro that have demonstrated inter-organelle exchange of lipids.