FEBS Letters 580 (2006) 2160–2165
Minireview
Endoplasmic reticulum: A metabolic compartment Miklo´s Csalaa, Ga´bor Ba´nhegyib, Angelo Benedettib,c,* a
Deptartment of Medical Chemistry, Semmelweis University and Endoplasmic Reticulum Research Group of the Hungarian Academy of Sciences, 1444 Budapest, Hungary b Department of Physiopathology, Experimental Medicine and Public Health, University of Siena, Viale Aldo Moro, 43100 Siena, Italy c Unit for Development of Biomedical Research, Santa Maria alle Scotte Hospital, 53100 Siena, Italy Received 3 February 2006; revised 7 March 2006; accepted 15 March 2006 Available online 29 March 2006 Edited by Felix Wieland
Abstract Several biochemical reactions and processes of cell biology are compartmentalized in the endoplasmic reticulum (ER). The view that the ER membrane is basically a scaffold for ER proteins, which is permeable to small molecules, is inconsistent with recent findings. The luminal micro-environment is characteristically different from the cytosol; its protein and glutathione thiols are remarkably more oxidized, and it contains a separate pyridine nucleotide pool. The substrate specificity and activity of certain luminal enzymes are dependent on selective transport of possible substrates and co-factors from the cytosol. Abundant biochemical, pharmacological, clinical and genetic data indicate that the barrier function of the lipid bilayer and specific transport activities in the membrane make the ER a separate metabolic compartment. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Endoplasmic reticulum; Membrane; Permeability; Transport; Compartment; Barrier; Latency; Specificity; Lumen
1. Introduction The endoplasmic reticulum (ER) is a continuous membrane network [1] in the cytosol. Its closed internal compartment, the ER lumen, can comprise about 10% of the total cell volume. Several metabolic pathways are compartmentalized in the ER. In particular, the active center of participating enzymes is localized in the lumen. These pathways are related to carbohydrate metabolism, biotransformation, steroid metabolism and protein processing [2]. The involved enzymes often receive their substrates and cofactors from, or release their products
* Corresponding author. Fax: +39 0577 234 009. E-mail address:
[email protected] (A. Benedetti).
Abbreviations: ER, endoplasmic reticulum; GSH, glutathione; GSSG, glutathione disulfide; GSD 1, glycogen storage disease type 1; CRD, cortisone reductase deficiency; G6P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; G6PT, glucose-6-phosphate transporter; H6PDH, hexose-6-phosphate dehydrogenase; 11bHSDH1, 11b-hydroxysteroid dehydrogenase type 1; MHC, major histocompatibility complex; RER, rough endoplasmic reticulum; TAP, transporter associated with antigen processing; UGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase
to, the cytosol. Therefore, passage of those compounds across the ER membrane is indispensable. There is still a debate whether the ER lumen can be considered as a separate metabolic compartment, or it is just a site in the cytosol for some enzymatic reactions. Because of the continuous inward and outward traffic of a large number of molecules, some scientists conclude that the ER membrane is permeable to all the small compounds, and acts only as a cheesecloth to keep the luminal proteins together. However, this view is contradicted by convincing experimental and clinical evidence indicating that the ER membrane forms a real barrier to several substances and that the trans-membrane transport is selective. In fact, the pattern of compounds that cannot enter or leave the lumen is just as important a determinant of the ER metabolism as the pattern of compounds that can. The characteristic micro-environment of the ER lumen could hardly been maintained if passive diffusion, driven by the gradients, effectively equalized the concentrations on the two sides of the membrane. The most important arguments for, and against, the general permeability of the ER membrane are summarized in Table 1. Which are the most relevant in vivo observations that support the barrier function of the ER membrane? The two best studied gradients across the ER membrane are related to the calcium homeostasis and the oxidative protein folding. Compared to the cytosol, the ER lumen has about four orders of magnitude higher level of free calcium ion [3] and nearly a hundred times lower ratio of glutathione (GSH) and oxidized glutathione disulfide (GSSG) [4]. Although the generation of these gradients is largely dependent on the poor permeability of the ER membrane to calcium ion and GSH, they are maintained or created by continuous active processes, such as pumping of calcium [3] and luminal oxidation of thiols [5], respectively. It is, therefore, arguable that high-capacity active processes surpass the velocity of passive trans-membrane fluxes. The strongest evidence for separation of the cytosolic and endoplasmic compartments were provided by genetic analysis of two ER-related human syndromes, namely the glycogen storage disease type 1 (GSD 1) and the cortisone reductase deficiency (CRD). The former proved that glucose 6-phosphate (G6P) is unable to enter the ER lumen, unless it is mediated by a specific transporter, while the latter revealed that the luminal cortisone reduction is dependent on local NADPH generation, because the cytosolic and the endoplasmic pyridine nucleotide pools are separated. GSD 1 refers to inborn deficiency of glucose 6phosphatase (G6Pase) activity, which causes a complex meta-
0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.03.050
M. Csala et al. / FEBS Letters 580 (2006) 2160–2165
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Table 1 Argument over a general permeability of the ER membrane Pros Membrane composition Low cholesterol and high protein content might increase leakiness by making the membrane structure less ordered [43] Translocon Transiently open transmembrane tunnel may allow non-selective fluxes [40,42] Size-dependence Chemical modification of intraluminal proteins by biotin-derivatives of different size up to 5 kDa indicate that compounds of a wide range of molecular weight have free access to the luminal compartment [44] Cons Selectivity The ER membrane is permeable to certain molecules (e.g. G6P [8,45], dehydroascorbate [26,27], FAD [5,25]) while impermeable to compounds of very similar size, structure, charge and polarity (e.g. other hexose phosphates [8,13], ascorbate [26,27], pyridine nucleotides [16,18,19], respectively) Exclusion Several intraluminal enzyme activities are nearly 100% latent in intact microsomes or in intact in situ ER [30,32,33] Entrapment Some compounds generated in the lumen accumulate at remarkable concentrations (e.g. GSSG [22,23], ascorbate [26], glucose [45], 6-phosphogluconate [9]). Concentration gradients can also be maintained by active transport (e.g. Ca2+). Calcium entrapment is enhanced by the intraluminal G6Pase activity [46] Inhibitors Transmembrane fluxes can be hampered by specific (e.g. chlorogenic acid derivatives in case of G6PT [47,48]) or general (e.g. 4,4 0 -diisothiocyanostilbene-2,2 0 -disulfonic acid [23,25,26,36,48]) transport inhibitors
bolic disorder including abnormal storage of glycogen in the liver [6]. Besides the defects of G6Pase enzyme itself (GSD 1a), mutations in a different gene were proven to cause similar metabolic disturbances combined with some additional symptoms [7] – a disease called GSD 1b. The protein encoded by this other gene turned out to be the glucose 6-phosphate transporter (G6PT), which is apparently needed for the access of the luminal G6Pase to its substrate [7–9]. CRD is based on insufficient reduction of cortisone to cortisol by 11b-hydroxysteroid dehydrogenase type 1 (11bHSDH1) in the ER lumen. The mutation analyses showed that this phenotype is caused by the combined defects of 11bHSDH1 and hexose 6-phosphate dehydrogenase (H6PDH) [10]. This finding indicates that the luminal generation of NADPH by H6PDH (rather than cytosolic NADPH production) drives the cortisone reduction in physiological conditions. This conclusion was validated by the results obtained in H6PDH knockout mice [11]. Hereafter this review summarizes the elucidated role of the ER membrane (as a barrier with selective transport) in the best studied metabolic systems of the ER.
2. G6Pase system Hydrolysis of G6P is the common ultimate step of glucose production from glycogen breakdown or gluconeogenesis in liver and kidney. The reaction is catalyzed by G6Pase, an integral membrane protein of the ER with intraluminal active center [8]. The system, therefore, theoretically comprises three transporters (for G6P, glucose and phosphate) associated functionally to the enzyme activity [8] (Fig. 1). One of the participating transporters, G6PT has been identified at molecular level [7,9]. Glucose and galactose transport across the ER membrane has been recently investigated in situ by using
genetically encoded fluorescence resonance energy transfer nanosensors [12]. Although the specific glucose transporter still remains to be identified, it was concluded that the remarkable differences in the kinetics strongly argue against the presence of a non-selective pore. Intact membrane barrier is an important determinant of the physiological characteristics of G6Pase. Permeabilization of microsomal vesicles only doubles the rate of G6P hydrolysis whilst it causes a 10–15-fold enhancement of the hydrolysis of mannose 6-phosphate and various other sugar phosphates, which become as good substrates of the enzyme as G6P [13]. Similar results were obtained using in situ permeabilized ER membranes [14,15]. The high substrate specificity of G6Pase in physiological conditions, therefore, relies on the selective transport of G6P into the lumen of ER and is not an intrinsic property of the enzyme. It is vitally important that the ER membrane separates the unselective G6Pase from the phosphoester intermediates of glycolysis/gluconeogenesis and amino sugar metabolism. The metabolic derangements in GSD 1a (G6Pase deficiency) and GSD 1b (G6PT deficiency) are indistinguishable and based in both cases on insufficient G6Pase activity [6]. This fact is evidently inconsistent with the theory of an un-specifically permeable ER membrane.
3. H6PDH and 11b-HSDH1 11bHSDH1 catalyzes reversible interconversion of cortisone and cortisol in the lumen of the ER (Fig. 1). The predominant in vivo direction is cortisone reduction, which is driven by a high luminal [NADPH]/[NADP+] ratio. The high ratio can be maintained by luminal dehydrogenases, such as H6PDH [9,16,17], because the permeability of the ER membrane to pyridine nucleotides is insignificant [18,19].
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Fig. 1. ER, a metabolic compartment. Certain luminal enzyme systems include transport activities importing their substrates and cofactors or exporting their products. Transporters may narrow the substrate specificity of the luminal catalytic subunits (e.g. G6Pase) [8] and/or determine the reaction rate (e.g. UGTs) [32]. The luminal localization of glucuronidation and de-glucuronidation necessitates translocation of substrates and glucuronides of various size and structure [30,34,35]. Separate, ER-specific redox of a pyridine nucleotide pool can be maintained by co-operating intraluminal enzyme activities (H6PDH, 11bHSDH) [10,16,17]. The protein facilitating G6P transport across the ER membrane has been identified [7]. Ovals and circles represent enzymes and transporters, respectively. GA, glucuronate; G6P, glucose-6-phosphate; G6PT, glucose-6-phosphate transporter; G6Pase, glucose-6-phosphatase; GUS, microsomal b-glucuronidase; H6PDH, hexose 6-phosphate dehydrogenase; 11bHSDH1, 11bhydroxysteroid dehydrogenase type 1; UGA, UDP-glucuronate; UGT UDP-glucuronosyltransferase; X-OH, aglycone (various); X-GA, aglyconeglucuronide (various).
It has been reported that extravesicular NADP+ and NADPH can penetrate the ER membrane in long incubations and that also the cytosolic [NADPH]/[NADP+] ratio can affect 11bHSDH1 activity [20]. Nevertheless, H6PDH knockout mice lack 11bHSDH1 mediated glucocorticoid generation [11], which proves that the enzyme cannot rely on cytosolic NADPH resources and a separate luminal pyridine nucleotide pool exists. It also clearly shows that the high luminal [NADPH]/[NADP+] ratio is dependent on the H6PDH activity and – consequently – on transport of G6P across the ER membrane (Fig. 1). In fact, substrate specificity of H6PDH is largely dependent on its localization in the ER. This 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 also has dual nucleotide specificity for NADP+ and NAD+. Nevertheless, under physiological conditions in the ER lumen the native substrates for H6PDH are believed to be G6P and NADP+ [21]. These findings indicate that the enzyme has no access to cytosolic NAD+, sugars and sugar phosphates, except G6P, which is transported by the specific G6PT [8].
4. 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 ones. This different [protein thiol]/[protein disulfide] ratio is also reflected by a different [GSH]/[GSSG] ratio, which is nearly 100:1 in the cytosol and about 1:1 in the ER lumen [4]. 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 isolated hepatic microsomes still contain GSH and GSSG [22] strongly suggested that the ER membrane represents a barrier for these molecules. Indeed, the ER membrane is impermeable to GSSG while GSH has a slow proteinmediated transport [23]. Therefore, locally oxidized GSH is entrapped in the form of GSSG in the lumen, contributing to the maintenance of the oxidizing environment in the compartment. Luminal thiol oxidation is facilitated by ascorbate (vitamin C) [24] or FAD [5,25]; so the physiological role of their transport has been proposed. ER membrane is selectively permeable to dehydroascorbate, the oxidized form of ascorbate [26,27]. Luminal reduction of dehydroascorbate to ascorbate is associated with thiol oxidation and leads to an ascorbate entrapment [28]. FAD uptake and a consequent thiol oxidation have also been found in yeast and liver microsomes [5,25]. 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 11bHSDH1 [16] and by direct transport measurements [19].
M. Csala et al. / FEBS Letters 580 (2006) 2160–2165
5. Glucuronidation The major second-phase reaction of hepatic biotransformation is glucuronidation that takes place in the ER (Fig. 1). Transfer of glucuronosyl group from UDP-glucuronate (UGA) to appropriate functional groups of the substrates is catalyzed by UDP-glucuronosyltransferases (UGTs). These enzymes are integral membrane proteins of the ER with their active center localized in the lumen [29]. 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 [30] and glucuronide export across the ER membrane. It has been demonstrated, using photoaffinity-labeling technique, that UGA but not UDP-glucose has access to the active center of UGTs in intact microsomal vesicles [31]. The high (more than 90%) latency of UGTs observed both in microsomal vesicles and isolated permeabilized hepatocytes indicates that transport processes (presumably UGA uptake) are rate limiting [32,33]. Activity of the intraluminal b-glucuronidase, a glucuronide-cleaving enzyme, is also limited by substrate (glucuronide) transport, although it has a smaller (approximately 40%) latency [34] (Fig. 1). Protein-mediated glucuronide transport across the ER membrane has been demonstrated [35], and competition for the transport has been found between glucuronides of similar size [36]. The pattern of interactions suggested the presence of multiple glucuronide transporters with overlapping specificities in the ER membrane [36].
6. The transporter associated with antigen processing (TAP) Beside G6PT, TAP peptide transporter – belonging to the superfamily of ABC transporters – is the only ER transporter characterized at molecular level. Oligopeptides (approx. 8–16 amino acids of length) produced by the proteasome in the cytosol are translocated by the heterodimer of TAP1 and TAP2 to the ER lumen, where they bind to 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. Loss of TAP function leads to an impairment of antigen presentation, as it is commonly observed in tumors and virus-infected cells that escape immune surveillance [37]. This condition clearly shows that basal permeability of the ER membrane does not allow the appearance of these small oligopeptides in the lumen.
7. Translocon and permeability of rough ER (RER) membrane Abundance of channels may theoretically contribute to a general permeability or leakiness of the ER membrane. This has been demonstrated in case of ribosome-bound translocon complex, which co-translationally imports nascent peptides into the RER lumen [38]. The average diameter of translocon ˚ , which is sufficiently wide to altunnel is approximately 20 A 2+ low transport of Ca [39] and small water-soluble molecules [40]. Therefore, the presence of translocon complexes might be responsible for higher permeability of rough versus smooth ER membrane [38].
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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 [41]. In fact, it has been argued that the BiP lock forms smaller barrier for uncharged polar molecules than for charged ones. Furthermore, dissociation of ribosomes from translocon complexes is delayed after the termination of protein synthesis. When a non-translating ribosome is associated to translocon complex, they form a transitional low-selectivity channel between the cytosol and the ER lumen [42].
8. Conclusion The proposed general, non-specific permeability of the ER membrane is clearly incompatible with several experimental findings and physiological or pathological observations. The collected data provide sufficient evidence for the presence of a barrier between the cytosol and the ER lumen. This barrier gives rise to generation of a characteristic micro-environment in the lumen with a higher Ca2+ concentration and with a more oxidized GSH redox-buffer. It also plays role in the modulation of certain ER-associated activities by limiting the accessibility of luminal enzymes to their substrates and – by this means – it contributes to their specificity as well. On the other hand, compared with the plasma membrane or some other cellular membranes, the ER membrane – especially the RER membrane – is relatively permeable, in terms of allowing detectable penetration of a number of investigated molecules. The huge difference between the diffusion rates of similar compounds, as well as the existence of substances apparently unable to permeate provide convincing evidence that lowspecificity transporters rather than a leaky lipid bi-layer are responsible for the phenomenon. Acknowledgments: OTKA (National Scientific Research Fund) F037484 and T048939, The Hungarian Ministries of Education (FKFP 0108/2001) and Welfare (ETT 090 and 613/2003) and The Italian Ministry of University and Research (Grant No. RBAU014PJA) provided support. The two research groups have a Bilateral Hungarian-Italian Intergovernmental S&T Cooperation Grant.
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