CHEMICAL BIOLOGY

Endoplasmic Reticulum: Topics in Chemical Biology

Gabor Banhegyi and Angelo Benedetti, Department of

Physiopathology, Experimental Medicine and Public Health, University of Siena, Siena, Italy

Miklos Csala, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary

doi: 10.1002/9780470152672.wecb152

Several pathways are compartmentalized in the endoplasmic reticulum (ER). These intraluminal activities require the passage of substrates, cofactors, and products through the ER membrane. The arguments for a general permeability of the ER membrane are contradicted by strong biochemical, pharmacological, clinical, and genetic evidence, which indicates that the lipid bilayer has a barrier function and that specific transport activities are needed in the membrane. Consequently, the ER lumen can be regarded as a separate metabolic compartment. This article overviews the best characterized intraluminal processes in which the compartmentation is important either by defining an intraluminal milieu, by limiting the rate of the reaction, by determining the specificity, or by creating a common substrate pool because of the colocalization.

The best known functions of the ER require a high membrane surface and/or a separate, specific microenvironment within the organelle. Although many enzymes hosted by the ER use its membranous structure only as a scaffold, others are compartmentalized within the ER; i.e., their active site is localized in the lumen. The activity of these enzymes usually is dependent on the special composition of the luminal compartment. The enzymes often receive their substrates and cofactors from or release their products to the cytosol; therefore, the transport of these compounds across the ER membrane is indispensable. This article focuses on this latter group of the ER enzymes, the functioning of which makes the ER a separate metabolic compartment of the eukaryotic cell.

Biologic Background

The ER is a continuous network of membranous tubular and lamellar structures in the cytosol (1, 2). The membranes that build up the organelle can constitute more than 95% of the total cellular membranes, and the total volume of the organelle can compose about 10% of cell volume, e.g., in hepatocytes. Although the ER is a single, spatially continuous compartment, it can be divided structurally and functionally into different subdomains. The ER forms contact sites beside the nuclear envelope with practically all the other organelles and the plasma membrane; these junctional regions might have specific composition and features, but they are restructured continuously to adapt the actual cellular requirements. The ER network can be divided into rough (RER) and smooth (SER) domains on the basis of the presence or absence of ribosomes on the outer surface of the membrane, respectively. Functional and morphological differences are linked: Whereas the ER is responsible for the synthesis, posttranslational modification, and folding of secretory and membrane proteins, the SER is responsible for lipid biosynthesis, biotransformation, and the production of small molecules to be secreted.

The ER hosts several enzymes: Some of them are facing toward the cytosol and use the ER membrane as a scaffold, e.g., cytochrome P450 isozymes, NADPH: cytochrome P450 reductase, or hydroxymethyl-glutaryl-CoA reductase. Others are compartmentalized in the ER; that is, their active center is localized in the lumen. The reactions catalyzed by these enzymes belong to various pathways of biochemistry and cell biology, such as carbohydrate and lipid metabolism, biotransformation, signaling, steroid metabolism, and protein processing. Table 1 presents a list of some important intraluminal enzymes. Usually, because their substrates derive from the cytosol, the corresponding membrane transport processes are required for their action.

Table 1. Some important activities compartmentalized within the ER lumen

Arguments for and against the general permeability of the ER membrane

It has been questioned whether the ER lumen can be considered as a separate metabolic compartment or whether the lumen is just a specific localization for some enzymatic reactions. The inward and outward traffic of a wide variety of molecules led some scientists to the conclusion that the ER membrane is permeable to any low-molecular-weight compounds and acts only as a molecular sieve to keep the luminal proteins together. Several arguments can support this hypothesis. The composition of the ER membrane is different from that of the plasma membrane; it has a low cholesterol and high protein content. The less-ordered membrane structure might increase leakiness for small molecules (3). The RER is abundant in translocon protein channels, which can allow the passage of ions and small-molecular-weight molecules (4-6). Other transiently open transmembrane channels might also allow nonselective fluxes. Moreover, a high number of structurally unrelated molecules, i.e., xenobiotics or biotin-derivatives of different size up to 5kDa (7), have free access to the luminal compartment as indicated by the chemical modifications of intraluminal proteins. However, this view is contradicted by convincing experimental and clinical evidence that indicates that the ER membrane forms a barrier to several substances and that the transmembrane transport is selective. In fact, the ER membrane is permeable selectively to certain molecules—e.g., glucose 6-phosphate (8, 9), dehydroascorbate (10, 11), or FAD (12, 13)—although it is impermeable to compounds of very similar size, structure, charge, and polarity—e.g., other hexose phosphates (8, 14), ascorbate (10, 11), or pyridine nucleotides (15-18), respectively. The selective permeability results in the exclusion and/or entrapment of metabolites from/in the lumen. A consistently observed feature of the intraluminal enzymes of the ER is the phenomenon of latency; i.e., the velocity of the reaction is low in case of intact ER membrane, which excludes the substrates from the lumen. The total enzymatic activity can be revealed on the destruction (permeabilization) of the membrane. Several intraluminal enzyme activities are nearly 100% latent in intact microsomes or in intact in situ ER (19-21). On the other hand, many compounds—generated locally in the lumen—accumulate at remarkable concentrations [e.g., glutathione disulfide (22, 23), ascorbate (10), glucose (9), or 6-phosphogluconate (24)]. The concentration gradient, maintained by active transport in case of Ca²⁺, is enhanced by coentrapment with inorganic phosphate generated by the intraluminal glucose 6-phosphatase activity (25). Finally, transmembrane fluxes can be hampered by specific [e.g., chlorogenic acid derivatives in case of glucose 6-phosphate translocase—G6PT (26, 27)] or general [e.g., 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (10, 13 , 23 , 27, 28)] transport inhibitors, which clearly indicates a protein-mediated transport. Summarizing these observations, the pattern of the compounds that cannot enter or leave the lumen is just as important a determinant of the ER metabolism as the pattern of the compounds that can enter or leave the lumen. The characteristic micro-environment of the ER lumen hardly could be maintained if the passive diffusion driven by the gradients equalized effectively the concentrations on both sides of the membrane.

In vivo evidences

Relevant in vivo observations support the hypothesis that the ER membrane has a barrier function. The two best studied gradients across the ER membrane are related to the calcium homeostasis and the oxidative protein folding. Compared with the cytosol, the ER lumen is characterized by a magnitude level of free calcium ion (29) that is four orders higher and nearly a hundred times lower than the ratio of glutathione (GSH) and oxidized glutathione disulfide (GSSG) (30). Although the generation of these gradients is dependent on largely the poor permeability of the ER membrane to calcium ion and glutathione, the gradients necessarily are created and maintained by continuous active processes, such as the pumping of calcium (29) and the luminal oxidation of thiols (12), respectively. Therefore, it can be also concluded that high-capacity active processes surpass the velocity of the passive transmembrane fluxes.

The strongest evidence for the separation of the cytosolic and endoplasmic compartments is provided by the genetic analyses of two ER-related human syndromes, namely glycogen storage disease type 1 (GSD 1) and cortisone reductase deficiency (CRD). GSD 1 refers to the congenital deficiency of glucose 6-phosphatase (G6Pase) activity, which causes a complex metabolic disorder, including the abnormal storage of glycogen in the liver (31). In addition to the defects of G6Pase enzyme (GSD 1a), the mutations in a different gene were proven to cause similar metabolic disturbances combined with some additional symptoms (32) —a disease called GSD 1b. The protein encoded by this other gene turned out to be the G6PT, which is needed to access the luminal G6Pase to its substrate (8, 24, 32). GSD 1b pathology was mimicked fully by G6PT knockout mice (33). The existence of GSD 1b proves that glucose 6-phosphate (G6P) cannot enter the ER lumen unless it is mediated by a specific transporter.

CRD is characterized by the insufficient reduction of cortisone to cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11PHSDH1) in the ER lumen. The analyses of the mutation suggest that the CRD phenotype is caused by the combined defects of 11βHSDH1 and hexose 6-phosphate dehydrogenase (H6PDH) (34). This finding indicates that the luminal generation of NADPH by H6PDH (rather than the cytosolic NADPH production) drives cortisone reduction in physiologic conditions. In other words, luminal cortisone reduction is dependent on local NADPH generation because the cytosolic and endoplasmic pyridine nucleotide pools are separated. Although recent investigations revealed that the combined mutations do not necessarily cause CRD (35, 36), the importance of the collaboration between the two enzymes was validated additionally by the results obtained in H6PDH knockout mice (37).

Another proof against the general permeability of the ER membrane and for the necessity of specific transporters is dervied from the field of immunology. TAP peptide transporters (TAP1 and TAP2), which were characterized at the molecular level, belong to the ABC transporter superfamily (38). They translocate oligopeptides (approximate length of 8-16 amino acid residues) produced by the proteasome in the cytosol to the ER lumen, where they bind to the major histocompatibility complex (MHC) Class I molecules. MHC-peptide complexes leave the ER by vesicular transport and reach the cell surface for recognition by cytotoxic T lymphocytes. The loss of the TAP function leads to the impairment of antigen presentation, as it is observed commonly in tumors and virus-infected cells that escape immune surveillance (39). This condition shows clearly that the basal permeability of the ER membrane does not permit the appearance of these small oligopeptides in the lumen.

Chemistry

In this section, the elucidated role of the ER membrane (as a barrier with selective transport) is summarized in the best studied metabolic systems of the ER.

Glucose 6-phosphatase system

The means of glucose production in liver and kidney is derived either from hydrolysis of G6P glycogen breakdown or gluco-neogenesis. The reaction is catalyzed by G6Pase, which is an integral membrane protein of the ER with an intraluminal active center (8). Theoretically, the system composes three transporters (for G6P, glucose, and phosphate) associated with the enzyme activity functionally (8). G6PT has been identified at the molecular level (24, 32), and the protein-mediated glucose transport across the ER membrane has been characterized recently (40).

The intact membrane barrier is an important determinant of the physiologic characteristics of G6Pase. Permeabilization of microsomal vesicles or in situ ER membranes (41, 42) only doubles the rate of G6P hydrolysis, and it causes a 1015-fold enhancement of the hydrolysis of mannose 6-phosphate and various other sugar phosphates by the same enzyme (14). It is important that the ER membrane separates the unselective G6Pase from intermediates of glycolysis/gluconeogenesis and amino sugar metabolism. However, the high substrate specificity of G6Pase in physiologic conditions is not an intrinsic property of the enzyme but relies on the selective transport of G6P into the lumen of ER.

GSD 1a (G6Pase deficiency) and GSD 1b (G6PT deficiency) cause the same metabolic derangements based on insufficient G6Pase activity (31, 33). This fact is inconsistent evidently with the theory of an unspecifically permeable ER membrane.

Hexose 6-phosphate dehydrogenase and 11β-hydroxysteroid dehydrogenase type 1

Interconversion of cortisone and cortisol catalyzed by 11βHSDH1 is fully reversible in vitro. The physiologic direction in vivo is cortisone reduction, which is driven by a high [NADPH]/[NADP⁺] ratio in the ER lumen. The high ratio can be maintained only by local NADP⁺ reduction catalyzed by certain luminal dehydrogenases, such as the H6PDH (15, 16, 24), because the permeability of the ER membrane to pyridine nucleotides is negligible (17, 18).

It has been reported that extravesicular NADP⁺ and NADPH can penetrate the ER membrane in long incubations and that the cytosolic [NADPH]/[NADP+] ratio can affect 11PHSDH1 activity (43). Nevertheless, the H6PDH knockout mice lack 11βHSDH1-mediated glucocorticoid generation (37), which proves that the enzyme cannot rely on cytosolic NADPH resources and that a separate luminal pyridine nucleotide pool exists. It also shows that clearly the high luminal [NADPH]/[NADP+] ratio is dependent on H6PDH activity.

G6P is transported to H6PDH from the cytosol across the ER membrane. In fact, similarly to G6Pase, the substrate specificity of H6PDH is dependent on largely its localization in the ER. The enzyme has dehydrogenase activity on various hexose 6-phosphates, such as G6P, galactose 6-phosphate, or 2-deoxyglucose 6-phosphate and on simple sugars such as glucose, and it has dual nucleotide specificity for NADP⁺ and NAD⁺. Nevertheless, under physiologic conditions in the ER lumen, the native substrates for H6PDH are believed to be G6P and NADP⁺ (44). These findings indicate that the enzyme has no access to the cytosolic NAD⁺, sugars, and sugar phosphates, except G6P, which is transported by the specific G6PT (8).

Oxidative protein folding and antioxidant metabolism

Luminal proteins and luminal domains of membrane proteins of the ER contain remarkably more disulfide bridges and less thiol groups than the cytosolic proteins. In accordance with this low [protein thiol]/[protein disulfide] ratio, the ER lumen shows also a characteristically low (about 1:1) [GSH]/[GSSG] ratio, which is nearly 100:1 in the cytosol (30). Such a high potential difference and concentration gradient would be hard to maintain if the membrane was permeable to both GSH and GSSG. The finding that indicates that isolated hepatic microsomes still contain GSH and GSSG (22) suggested strongly that the ER membrane represents a barrier for these molecules. Indeed, the membrane is impermeable to GSSG, whereas GSH has a slow protein-mediated transport (23). Therefore, the oxidizing environment in the compartment can be maintained by local oxidation of GSH that yields GSSG, which is entrapped in the lumen.

Luminal thiol oxidation is facilitated by ascorbate (vitamin C) (45) or FAD (12, 13), so the physiologic role of their transport has been proposed. ER membrane is permeable selectively to dehydroascorbate, the oxidized form of ascorbate (10, 11). Luminal reduction of dehydroascorbate to ascorbate is associated with thiol oxidation and leads to ascorbate entrapment (46).

FAD uptake and a consequent thiol oxidation have also been found in yeast and in liver microsomes (12, 13). In contrast to FAD, pyridine nucleotides—NADP(H), NAD(H)—of similar size and structure cannot enter the ER lumen at a significant rate, which is indicated by the high latency of intraluminal H6PDH and 11βHSDH1 (15) and by direct transport measurements (18).

Glucuronidation

The quantitatively most significant second-phase reaction of hepatic biotransformation is glucuronidation that takes place in the ER. The transfer of glucuronosyl group from UDP-glucuronate (UGA) to appropriate functional groups of the substrates is catalyzed by UDP-glucuronosyltranferases (UGTs). These enzymes are integral membrane proteins of the ER with their active center localized in the lumen (47). UGA is synthesized in the cytosol, and the produced glucuronides are pumped out of the cell by plasma membrane transporters. Thus, conjugation with glucuronate requires UGA import (19) and glucuronide export across the ER membrane. It has been demonstrated, using a photoaffinity-labeling technique, that UGA, but not UDP-glucose, has access to the active center of UGTs in intact microsomal vesicles (48). The high latency (more than 90%) of UGTs observed in both microsomal vesicles and isolated permeabilized hepatocytes indicates that the transport processes (presumably UGA uptake) are rate limiting (20, 21). Activity of the luminal β-glucuronidase, a glucuronide-cleaving enzyme, is also limited by substrate (glucuronide) transport, although it has moderate (approximately 40%) latency (49).

A protein-mediated glucuronide transport across the ER membrane has been demonstrated (50), and a competition for the transport has been found between glucuronides of similar size (28). The pattern of interactions suggested the presence of multiple glucuronide transporters with overlapping specificities in the ER membrane (28).

Translocon and permeability of (R)Er membrane

Abundance of channels may contribute theoretically to a general permeability or leakiness of the ER membrane, which has been demonstrated in case of the ribosome-bound translocon complex, which cotranslationally imports nascent peptides into the RER lumen (51). The average diameter of the translocon tunnel is approximately 20 A, which is wide enough to allow the transport of Ca²⁺ (52) and small water-soluble molecules (4-6). Therefore, the presence of translocon complexes might be responsible for the higher permeability of the RER membrane versus the SER membrane.

Unspecific permeability is prevented in the translationally active translocon because the tunnel is occupied by the peptide chain being polymerized. Similarly, the pore is blocked by BiP proteins, prominent intraluminal chaperones, after dissociation of the ribosome from the complex (53). In fact, it has been argued that the BiP locks form a smaller barrier for uncharged polar molecules than for charged ones. Furthermore, the dissociation of ribosomes from translocon complexes is delayed after the termination of protein synthesis. When a nontranslating ribosome is associated with the translocon complex, they form a transitional low-selectivity channel between the cytosol and the ER lumen (5).

Chemical Tools and Techniques

In vivo studies

Measurement of the concentrations in the cytosolic and luminal compartments in vivo has been achieved in the case of calcium ion but remains a merely theoretical possibility for most organic molecules except glutathione (54). In fact, a remarkable transmembrane gradient has been detected in case of both Ca²⁺ and glutathione, which supports the barrier function of the ER membrane. The maintenance of high concentration differences would require continuously intensive pump activities and lead to unreasonable heat generation if the membrane is highly permeable.

Studies in cellular systems

The potential role of transport across the ER membrane was investigated in cells by using selective and general membrane permeabilizing toxins, antibiotics, or detergents. Intact in situ ER can be exposed to the incubation medium in cells whose plasma membrane has been permeabilized selectively. Alternatively, the intraluminal enzymes can be directly exposed by general (including plasma and ER membranes) permeabilization. Experiments using these models revealed that the transport of substrates or cofactors across the ER membrane is rate limiting for G6Pase (42), UGT (20), and β-glucuronidase (49) activities.

The transport of exogenous compounds has been investigated recently by determining the amount of modified intraluminal proteins upon addition of nonphysiologic biotin-derivatives capable of chemical protein modification (7). The ER membrane has been shown to be permeable to three different biotin-derivatives, and it has been concluded that it does not form a barrier to small molecules; that is, either the lipid bilayer or the integral membrane proteins allow their diffusion unspecifically.

Studies in subcellular systems

Isolated ER-derived vesicles (microsomes) maintain their original orientation [i.e., their intravesicular surface corresponds to the intraluminal side of the ER membrane (55, 56)] and keep some of their low-molecular-weight luminal components during the long-lasting preparation procedure. For example, both oxidized and reduced forms of glutathione (22) and of pyridine nucleotides (17) are present in liver microsomes, which suggests strongly that the ER membrane acts as a barrier to these molecules.

Microsomes are used widely for the determination of “isotope space” and for in vitro transport assays, such as “rapid filtration” and “light scattering” measurements. Isotope spaces reveal the distribution of the studied compound between intra- and extravesicular water compartments; hence, the measurements provide information on the permeability but not on the transport kinetics. The total water space and the extravesicular space can be measured using radiolabeled analogs of water or a completely impermeable compound, respectively (57). Rapid filtration is based on the quick separation of vesicle-associated molecules from the medium and on the calculation of the intraluminal content after various incubation periods. This method allows the application of physiologic concentrations but offers a limited time resolution (58). On the contrary, the light scattering technique requires high concentrations but provides a real-time monitoring of traffic. This indirect detection takes advantage of the transport-associated osmotic shrinkage and the swelling of microsomal vesicles (58). The permeability to several molecules of a wide size-range has been compared with these methods, and very slow permeation, or complete impermeability, has been demonstrated in certain cases.

A sophisticated approach using active site-directed photoaffinity substrate analogs has also been applied to study the inward transport in isolated microsomal vesicles. These experiments provided convincing and elegant evidence for the translocation of UDP-glucuronate (48) and of FAD (12) by photo incorporation of the probes into the luminally oriented enzymes in intact microsomes. The drawback of the method is that it is not suitable to determine of the rate or capacity of transport.

Enzyme latency is an experimental manifestation of compart- mentation, which means that the activity of certain intraluminal enzymes is increased remarkably when the membrane is perme- abilized either by detergents or by channel-forming antibiotics (e.g., alamethicin). It is based on the rate-limiting transport of substrates and/or cofactors across the intact ER membrane. Some activities in the ER are more than 90% latent; i.e., they increase more than 10-fold during permeabilization (20, 21).

Conclusion

The ER membrane represents a real barrier between the cytosol and the lumen for water-soluble, charged small molecules. Although this barrier function is compromised at a certain extent by the existence of nonspecific pores (e.g., the translocon protein channel), the ER lumen can be considered still as a separate metabolic compartment. This barrier generates a characteristic microenvironment in the lumen with a higher Ca²⁺ concentration and with a more oxidized glutathione redox-buffer, both of which are required for the proper functioning of luminal processes, including the oxidative protein folding. It also plays a role in the modulation of certain ER-associated activities either by limiting the accessibility of luminal enzymes to their substrates or by ensuring their specificity as well.

Although several intraluminal activities have been explored in the ER, the identification of the proteins that participate in transport processes across the ER membrane lags behind. In comparison with other organelles, the transporters of the ER are less known. The reason might be the technical difficulty of the ER transport measurements and of the purification and reconstruction approach. The fact that the ER is a eukaryotic organelle that has no obvious relationship with any bacterial ancestor makes the in silico approach for the identification of transporter genes less fruitful. Moreover, it cannot be excluded that ER transport is facilitated by only a few proteins of low ligand-specificity. Experimental findings can support this possibility. However, the identification of new ER transporters on the basis of the ER proteome (59) will lead to a remarkable development in the better understanding of the biochemistry of the ER lumen.

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Further Reading

Benedetti A, Banhegyi G Burchell A. eds. Endoplasmic Reticulum: A Metabolic Compartment. 2005. IOS Press, Amsterdam, The Netherlands.

Borgese N, Harris JR. eds. Subcellular Biochemistry, 21. Endoplasmic Reticulum. 1993. Plenum Press, New York.

Csala M, Banhegyi G, Benedetti A. Endoplasmic reticulum: a metabolic compartment. FEBS Lett. 2006; 580:2160-2165.

Csala M, Marcolongo P, Lizak B, Sanesi S, Margittai E, Fulceri R, Magyar JE, Benedetti A, Banhegyi G. Transport and transporters in the endoplasmic reticulum. Biochim. Biophys. Acta 2007; 1768:1325-1341.

Banhegyi G, Benedetti A, Csala M, Mandl J. Stress on redox. FEBS Lett. 2007; 581:3634-3640.

UNSW Cell Biology. Endoplasmic Reticulum. Available at: http:// cellbiology.med.unsw. edu.au/units/components/er.htm

See Also

Compartment

Metabolic Diseases, Chemical Biology of

Metabolism, Cellular Organization of

Oxidative Post-Translational Modifications

Small Molecule Transport