Which of the following correctly states the functions of the rough endoplasmic reticulum?

Secretory and membrane proteins are synthesized in association with the rough ER

Transmembrane proteins are composed of hydrophobic domains that are embedded within the phospholipid bilayer and hydrophilic domains that are exposed at the intracellular and extracellular surfaces (seepp. 13–15). These proteins do not “flip” through the membrane. How, then, do intrinsic membrane proteins overcome the enormous energetic barriers that should logically prevent them from getting inserted into the membrane in the first place?

The cell has developed several schemes to address this problem. Mammalian cells have at least three different membrane insertion pathways, each associated with specific organelles. The first two are mechanisms for inserting membrane proteins into peroxisomes and mitochondria (seepp. 30–32). The third mechanism inserts membrane proteins destined for delivery to the plasma membrane and to the membranes of organelles (the endomembranous system) other than the peroxisome and mitochondrion. This same mechanism is involved in the biogenesis of essentially all proteins that mammalian cells secrete and is the focus of the following discussion.

The critical work in this field centered on studies of the rough ER. The membrane of therough ER (seepp. 20–21) is notable for the presence of numerous ribosomes that are bound to its cytosol-facing surface. Although all nucleated mammalian cells have at least some rough ER, cells that produce large quantities of secretory proteins—such as the exocrine cells of the pancreas, which function as factories for digestive enzymes (seepp. 879–881)—are endowed with an abundance of rough ER. Roughly half of the cytoplasmic space in an exocrine pancreatic acinar cell is occupied by rough ER.

In early experiments exploring cell-fractionation techniques, membranes that were derived from the rough ER were separated from the other membranous and cytoplasmic components of pancreatic acinar cells. The mRNAs associated with rough ER membranes were isolated and the proteins they encoded were synthesized by in vitro translation. Analysis of the resultant polypeptides revealed that they included the cell's entire repertoire ofsecretory proteins. It is now appreciated that the mRNA associated with the ER also encodes the cell's entire repertoire ofmembrane proteins, with the exception of those destined for either the peroxisome or the mitochondrion. When the same experiment was performed with mRNAs isolated from ribosomes that are freely distributed throughout the cytoplasm, the products were notsecretory proteins but rather the solublecytosolic proteins. Later work showed that the ribosomes bound to the ER are biochemically identical to and in equilibrium with those that are free in the cytosol. Therefore, a ribosome's subcellular localization—that is, whether it is free in the cytosol or bound to the rough ER—is somehow dictated by the mRNA that the ribosome is currently translating. A ribosome that is involved in assembling a secretory or membrane protein will associate with the membrane of the rough ER, whereas the same ribosome will be free in the cytosol when it is producing cytosolic proteins. Clearly, somelocalization signal that resides in the mRNA or in the protein that is being synthesized must tell the ribosome what kind of protein is being produced and where in the cell that production should occur.

Endoplasmic Reticulum, Ribosomes, and Golgi Apparatus

Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and Golgi complexes are abundant in mammalian hepatocytes (see Figs. 1-15 and 1-16).1,2,51,58 Their functions are related mainly to the synthesis and conjugation of proteins, metabolism of lipids and steroids, detoxification and metabolism of drugs, and breakdown of glycogen. The endoplasmic reticulum forms a continuous three-dimensional network of tubules, vesicles, and lamellae. Almost 60% of the endoplasmic reticulum has ribosomes attached to its cytoplasmic surface and is known as the RER. The remaining 40% constitutes the SER, which lacks a coating of ribosomes. The membranes of the endoplasmic reticulum are between 5 nm and 8 nm thick. The lumen of the RER is approximately 20 nm to 30 nm wide, whereas that of the SER is larger (30-60 nm). The morphologic characteristics and amount of the endoplasmic reticulum vary in the different zones of the liver lobule.

RER is arranged in aggregates of flat cisternae that may be found throughout the cytoplasm. It is more frequently distributed in the perinuclear, pericanalicular, and subbasilar regions of hepatocytes, and it is more abundant in periportal cells than in centrilobular cells.59 The numerous attached membrane-bound ribosomes consist of a large and a small subunit, with the large subunit attached to the RER. Free ribosomes and polyribosomes are also present within the hepatocyte cytoplasm. Ribosomes contain RNA and ribosomal proteins and play a key role in the synthesis of proteins, particularly those destined for secretion or for delivery to intracellular membrane compartments or the plasma membrane. Vesicles containing these proteins are directed to the proximate (cis) cisternae of the Golgi apparatus, for further processing.

SER is less common and has a more complex arrangement than RER.51 It is usually much more abundant in centrilobular than in periportal hepatocytes59,60; the high content of heme-containing cytochromes lends a darker pigmentation to the centrilobular region of the lobule, as is evident on visual inspection of the cut surface of the liver. The matrix within the SER tubules is usually slightly more electron-dense than the surrounding cytoplasm. SER membranes are irregular in size and present a tortuous course. They may be tubular or vesicular in structure with a width of 20 nm to 40 nm. SER is mainly distributed near the periphery of the cell. It is often in close relation to RER and Golgi membranes, as well as to glycogen inclusions.51

The ER is not the only site of protein synthesis in hepatocytes. Abundant free ribosomes in the cytoplasm participate in the synthesis of some proteins that will be secreted but synthesize essentially all of the structural proteins for the hepatocyte. Proteins that are to remain within the cytoplasm or are destined to enter the nucleus, peroxisomes, or mitochondria are completely synthesized by free ribosomes.

The Golgi complex is a three-dimensional structure in hepatocytes, characteristically consisting of a stack of four to six parallel cisternae, often with dilated bulbous ends containing electron-dense material.10,51,58 Multiple Golgi complexes exist in each hepatic parenchymal cell, generally distributed near the nucleus. This structure shows a convex or proximal portion facing the nucleus and the endoplasmic reticulum (cis-Golgi), where small vesicles transfer proteins from the endoplasmic reticulum to the Golgi, and a concave part (trans-Golgi), which connects with a post-Golgi trans-Golgi network that directs proteins towards their final destinations: to organelle membranes of the cell, the plasma membrane, or for secretion. The cisternae may be up to 1 µm in diameter with a lumen that is 30 nm wide. The Golgi complex is capable of rapid and reversible structural reorganization into a tubuloglomerular network, while maintaining its biosynthetic capabilities.61 With the SER, RER, lysosomes, other intermediate organelle compartments, and even the nuclear and mitochondrial envelope membranes, the Golgi is an integral part of the complex intracellular organelle network involving vesicular trafficking that enables uptake, sorting, degradation, biosynthesis, trafficking, and/or secretion of cellular proteins and lipids.60-63

Rough endoplasmic reticulum

The rough endoplasmic reticulum is a site of protein synthesis; its cytosolic surface is studded with ribosomes (Fig. 1.5B). Ribosomes only bind to the endoplasmic reticulum when proteins targeted for secretion begin to be synthesized. Most proteins pass through its membranes and accumulate within its cisternae, although some integral membrane proteins, e.g. plasma membrane receptors, are inserted into the rough endoplasmic reticulum membrane, where they remain. After passage from the rough endoplasmic reticulum, proteins remain in membrane-bound cytoplasmic organelles such as lysosomes, become incorporated into new plasma membrane, or are secreted by the cell. Some carbohydrates are also synthesized by enzymes within the cavities of the rough endoplasmic reticulum and may be attached to newly formed protein (glycosylation). Vesicles are budded off from the rough endoplasmic reticulum for transport to the Golgi as part of the protein-targeting mechanism of the cell

Endoplasmic Reticulum, Ribosomes, and Golgi Apparatus

Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and Golgi complex are abundant in mammalian hepatocytes (see Figs. 1-13 and 1-14).1,2,24,27 Their functions are related mainly to the synthesis and conjugation of proteins, metabolism of lipids and steroids, detoxification and metabolism of drugs, and breakdown of glycogen. The endoplasmic reticulum forms a continuous three-dimensional network of tubules, vesicles, and lamellae. Almost 60% of the endoplasmic reticulum has ribosomes attached to its cytoplasmic surface and is known as the RER. The remaining 40% constitutes the SER, which lacks a coating of ribosomes. The membranes of the endoplasmic reticulum are 5 to 8 nm thick. The lumen of the RER is approximately 20 to 30 nm wide, whereas that of the SER is larger (30 to 60 nm). The morphologic characteristics and amount of the endoplasmic reticulum may vary in the different zones of the liver lobule.

RER is arranged in aggregates of flat cisternae that may be found throughout the cytoplasm. It is more frequently distributed in the perinuclear, pericanalicular, and vascular regions of hepatocytes, and it is more abundant in periportal cells than in centrilobular cells.28 The numerous attached membrane-bound ribosomes consist of a large and a small subunit, with the large subunit attached to the RER. Free ribosomes and polyribosomes are also present within the hepatocyte cytoplasm. Ribosomes contain RNA and ribosomal proteins and play a key role in the synthesis of proteins.

SER is less common and has a more complex arrangement than RER.24 It is usually much more abundant in centrilobular than in periportal hepatocytes.28,29 The cytoplasm within the SER tubules is usually slightly more electron-dense than the surrounding cytoplasm. SER membranes are irregular in size and present a tortuous course. They may be tubular or vesicular in structure, with a width of 20 to 40 nm. SER is mainly distributed near the periphery of the cell. It is often in close relation to RER and Golgi membranes, as well as to glycogen inclusions.24

The ER is not the only site of protein synthesis in hepatocytes. Abundant free ribosomes in the cytoplasm participate in the synthesis of some proteins that will be secreted, but especially of all structural proteins for the hepatocyte. Messages encoding proteins that are to remain within the cytoplasm or are destined to enter the nucleus, peroxisomes, or mitochondria are completely synthesized by free ribosomes.

The Golgi complex is a three-dimensional structure in hepatocytes consisting of numerous membranes and vacuoles.8,24,27 Multiple Golgi complexes exist in each hepatic parenchymal cell. Whether or not these complexes are interconnected (functionally forming a single large organelle) is uncertain. The Golgi generally is distributed near the bile canaliculus or nucleus. The Golgi apparatus presents a characteristic heterogeneity. It is usually formed by a stack of four to six parallel cisternae, often with dilated bulbous ends containing electron-dense material. The cisternae may be up to 1 µm in diameter with a lumen that is 30 nm wide. This structure shows a convex or proximal portion facing the nucleus and the endoplasmic reticulum (cis Golgi), where small vesicles transfer proteins from the endoplasmic reticulum, and a concave part (trans Golgi), where vesicles and vacuoles (secretory granules) originate to transport the contained secretory proteins to the plasma membrane for discharge into the space of Disse. Both cis and trans Golgi are connected by means of the medial Golgi. The latter is the intermediate station between endoplasmic reticulum and Golgi products, such as secretory granules or secondary lysosomes (GERL). This arrangement of Golgi stacks corresponds to its morphofunctional polarization related to the pathway of protein passage through this structure. Proteins in fact enter via the cis Golgi, pass through the medial Golgi, and leave this structure via the exit pole (trans Golgi). Two main types of secretory vesicle can be identified within the Golgi apparatus: smaller presecretory granules of 50-nm diameter and larger secretory granules 400 to 600 nm in diameter containing proteins such as very low-density lipoproteins.30

Collagen synthesis begins in the rough endoplasmic reticulum (RER)

The steps in the synthesis of most fibrillary collagens are similar and involve the constitutive secretory pathway used by cells. Collagen α chains are synthesized in the RER as long precursors, pro-α chains, or preprocollagen molecules, which then undergo extensive modifications in the Golgi apparatus and extracellular space (Fig. 19.3). Preprocollagen is synthesized initially with a hydrophobic signal sequence that facilitates binding of ribosomes to the endoplasmic reticulum. Posttranslational modification of the protein begins with removal of the signal peptide in the ER, yieldingprocollagen. Three different hydroxylases then add hydroxyl groups to proline and lysine residues, forming 3- and 4-hydroxyprolines and δ-hydroxylysine. These hydroxylases require ascorbate (vitamin C) as a cofactor (Fig. 19.3). Vitamin C deficiency leads toscurvy as a result of alterations in collagen synthesis and crosslinking.

O-linked glycosylation occurs by the addition of galactosyl residues to hydroxylysine by galactosyl transferase; a disaccharide is formed by the addition of glucose to galactosyl hydroxylysine by a glucosyl transferase (Fig. 19.3). These enzymes have strict substrate specificity for hydroxylysine or galactosyl hydroxylysine, and they glycosylate only those peptide sequences that are in noncollagenous domains.N-linked glycosylation also occurs on specific asparagine residues in nonfibrillar domains. The nonfibrillar collagens, with a greater extent of nonhelical domains, are more highly glycosylated than fibrillar collagens. Thus the extent of glycosylation may influence fibril structure, interrupting fibril formation and promoting the interchain interactions required for a meshwork structure. Intra- and interchain disulfide bonds are formed in theC-terminal domains by a protein disulfide isomerase, facilitating the association and folding of peptide chains into a triple helix (Fig. 19.3). At this stage, theprocollagen is still soluble and contains additional nonhelical extensions at itsN- andC-terminals.

3.4 Ribosome-binding proteins

Rough ER contains two integral membrane proteins, ribophorins I and II, that have molecular masses of 65 kDa and 63 kDa, respectively. These proteins remain attached to ribosomes when the ER membrane is dissolved with detergent and they can be crosslinked to ribosomes by chemical reagents. It has been suggested that ribophorins provide the attachment site for the large subunit of the ribosome to the ER. Ribophorins I and II are type I membrane proteins with 150 and 70 amino acids, respectively, in the cytosol. Proteolytic treatment of microsomes, which prevents ribosome binding, does not, however, affect the structure of the ribophorins. Ribophorins cannot therefore be the major site of attachment for ribosomes to the ER. Ribophorins are known to be important subunits of the oligosaccharyl transferase enzyme (see below).

Sec61p is also a ribosome-binding protein and the Sec61p complex is likely the major ribosome binding site [21]. The Sec61p complex remains tightly associated with the ribosome after detergent solubilization of the ER membrane. Reconstitution studies [22,23] indicate that Sec61p complex is sufficient to translocate nascent chains across the ER membrane and that Sec61p is also required for the correct insertion of all types of single span membrane proteins into the ER. The ribosome provides a tight seal on the cytosolic side of the translocon ensuring a unidirectional passage of the nascent chain into the ER lumen. Protease digestion experiments have shown that the carboxyl-terminal 70 amino acids are protected by the ribosome and the translocon [24]. Since about 30 residues are protected by the ribosome alone, this suggests that an equal number of amino acids are sequestered within the translocon at any time. Synthesis of the polypeptide may provide some of the energy necessary for translocation through an aqueous channel. When a stop codon is reached, translation stops and the ribosome dissociates into subunits. This would leave a 30 amino acid carboxyl-terminal tail on the cytosolic side of the ER membrane. Protein folding within the lumen of the ER or the translocon itself may drive completion of translocation. Release of the nascent chain into the lumen of the ER would result in closure of the translocon.

Function of the OI osteoblast

The rough endoplasmic reticulum of OI fibroblasts and osteoblasts is grossly dilated [205] and the secretion of fully formed but mutant procollagen is impaired [206,207]. The role that the hsp47 chaperone protein plays in determining the trafficking of normal and mutant molecules within these cells is believed to be important in detecting the mutant collagen chains and eliciting a cellular mechanism to prevent their secretion [208]. In fact, gene knockout of the hsp47 protein is an embryonic lethal in which an abnormal type of collagen accumulates [209], suggesting that this chaperone protein plays an essential role in selecting for correctly assembled collagen molecules [210]. The retention of the mutant procollagen molecule also leads to posttranslational overmodification of the lysine residues in the helical domain that may further affect the quality of fibril formation.

In vitro studies of osteoblasts derived from OI humans [209,210] or OIM mice [211] show diminished markers of osteoblastic differentiation, as well as a reduced rate of cell proliferation. If this property of the OI osteoblast persists in vivo, it may be a secondary contributor to the severity of bone disease. Not only is there an impairment in the quantity or quality of the matrix that is produced, but the number of differentiated osteoblasts capable of making a mineralized matrix may also be reduced. The mechanism for diminished osteoblast proliferation and differentiation could be a direct consequence of the retained procollagen molecules with the distended rough endoplasmic reticulum. It may reflect an indirect effect of the quality or quantity of the extracellular matrix made by the preosteoblastic cell that is necessary for osteoblast differentiation [306,307]. Possibly, the high rate of bone turnover characteristic of this disease may lead to exhaustion and/or premature senescence of stem cells capable of generating vigorous osteoblastic cells in vitro, which if present in intact bone will further contribute to the severity of the bone disease, particularly in elderly subjects with OI.

Function of the OI Osteoblast

The rough endoplasmic reticulum of OI fibroblasts and osteoblasts is grossly dilated [200] and the secretion of fully formed but mutant procollagen is impaired [201,202]. The role that the HSP47 chaperone protein plays in determining the trafficking of normal and mutant molecules within these cells is believed to be important in detecting the mutant collagen chains and eliciting a cellular mechanism to prevent their secretion [203]. Recently mutations in SERPINH1 encoding HSP47 have been identified in patients with recessive severe OI [79]. In fact, gene knockout of the HSP47 protein is embryonic lethal. An abnormal type of collagen accumulates [204], suggesting that this chaperone protein plays an essential role in selecting for correctly assembled collagen molecules [205]. The retention of the mutant procollagen molecule also leads to post-translational overmodification of the lysine residues in the helical domain that may further affect the quality of fibril formation.

In vitro studies of osteoblasts derived from OI humans [204,205] or Oim mice [206] show diminished markers of osteoblastic differentiation, as well as a reduced rate of cell proliferation. If this property of the OI osteoblast persists in vivo, it may be a secondary contributor to the severity of bone disease. Not only is there an impairment in the quantity or quality of the matrix that is produced, but the number of differentiated osteoblasts capable of making a mineralized matrix may also be reduced. The mechanism for diminished osteoblast proliferation and differentiation could be a direct consequence of the retained procollagen molecules with the distended rough endoplasmic reticulum. It may reflect an indirect effect of the quality or quantity of the extracellular matrix made by the preosteoblastic cell that is necessary for osteoblast differentiation [207,208]. Possibly, the high rate of bone turnover characteristic of this disease may lead to exhaustion and/or premature senescence of stem cells capable of generating vigorous osteoblastic cells in vitro, which if present in intact bone will further contribute to the severity of the bone disease, particularly in elderly subjects with OI.

Endoplasmic reticulum stress, and unfolded protein response (UPR)

The endoplasmic reticulum (ER) (rough and smooth) consists of a complex network of closely related cisternae, sacs, and tubules, and a continuous network connects it into the nuclear membrane [98–100]. This major organelle is involved in the synthesis, folding, posttranslational modification, secretion, and transfer of approximately one-third of all proteins to a defined destination [101]. As a quality control gate, ER responds to the cellular stress by ER chaperones including GRP78, GRP94, and GRP18. The glucose-regulated protein (GRP) detects the presence of unfolded or misfolded proteins [102, 103]. The constant interaction of ER with the nucleus, mitochondria, and the Golgi apparatus makes it a key regulator in normal cell function [104]. Various internal or external stressors, including hypoxia, starvation, infection, calcium homeostasis, and/or disruption of cytotoxic compounds, directly or indirectly lead to the instability of the ER microenvironment and abnormal protein accumulation in the ER lumen commonly referred to as ER stress [105, 106]. In this condition, the normal balance between protein biosynthesis and normal protein folding in the ER is perturbed. Depending on the intensity and exposure time of the stress, adaptive defense systems are triggered by various compensation mechanisms such as the unfolded protein response (UPR), endoplasmic reticulum-associated degradation (ERAD), and autophagy [107, 108]. Cell death occurs when the adaptive pathway fails to compensate for the ER normal function [109, 110]. Activation of UPR in endoplasmic reticulum stress enhances normal protein folding by expression of the essential genes in protein folding, secretion, and quality control. Not only UPR regulates ER function, but also modulates the mitochondrial biology, lipid metabolism, and intracellular apoptotic pathways [111]. Nowadays, UPR is not only considered as a protein folding pathway [1] but new functions have been recognized, including lipid metabolism, energy control, and inflammation [112].

The UPR is an important target in the treatment of diabetes, cancers, immune diseases, neurodegenerative disorders, cataracts, pulmonary fibrosis, cardiomyopathy, atherosclerosis, ischemia, nephrotic syndrome, nonalcoholic steatosis, and psoriasis [113]. Nowadays, ER stress, UPR and other cell homeostasis pathways have attracted great attention in the field of cancer therapy. The ER stress response is regularly monitored by three specific UPR sensors: inositol requires enzyme 1 (IRE-1α and IRE-1β), protein kinase RNA-like ER kinase (PERK), and activated transcription factor 6 (ATF6α and ATF6β subtypes). These sensors are located on the outer membrane of the ER but are connected to GRP78 during an inactive state. Depending on the cell type, the stimuli, duration, and intensity of the stress, UPR may cause cell adaptation or cell death [105, 114].

GRP78 as a main regulator of the ER stress promotes the assembly, folding, and translocation of newly synthesized nascent proteins. It can inhibit protein accumulation and remove unfolded/misfolded proteins through the ER-related degradation (ERAD) process [114, 115]. GRP78 also regulates calcium homeostasis by ER-mitochondrial calcium cross talk [116].

In ER stress status, activated PERK dissociates from GRP78 and phosphorylates eukaryotic initiation factor 2 (eIF2α) to reduce the pressure on the ER. Dissociated PERK then suppresses the translation of excess cytoplasmic mRNA and prevents the newly synthesized proteins from entering the ER [115, 117, 118]. Phosphorylated eIF2α interferes with the translation of intracellular proteins, except for some important transcription factors such as activated transcription factors (ATF4) [119, 120]. Moreover, activated IRE-1 affects the X-box-binding protein (XBP-1) mRNA transcription factor through the endonuclease activity. Spliced and activated XBP-1 develops the protein folding capacity of ER by regulating the expression of several genes involved in ER quality control, ERAD, autophagy, and apoptosis [121]. Finally, ATF6 is separated from GRP78 and is transported to the Golgi organelle. The transported ATF6 is then activated by regulated intramembrane proteolysis (RIP) by site 1 and site 2 (SP) proteases. The cytoplasmic domain (50 kDa) of the activated ATF6 translocates to the nucleus [122, 123] and increases the levels of ER chaperones, XBP-1, and protein folding capacity. The entire cascade of UPR pathway is presented in Fig.5.

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