Science Wiki

The rough endoplasmic reticulum (abbreviated RER or rough ER) is a region of the endoplasmic reticulum, a membrane-bound organelle found in nearly all eukaryotic cells. It is a continuous network of flattened sacs (cisternae) and tubules that surrounds a single lumen. The RER is called "rough" because its cytosolic surface is studded with ribosomes, which give it a granular appearance in the electron microscope. It is the site of synthesis of most secretory proteins, of proteins that are inserted into the membranes of the secretory pathway and the plasma membrane, and of proteins that are delivered to the lumen of the ER, the Golgi apparatus, and lysosomes. It also takes part in the folding, modification, and quality control of these proteins.

The RER is not a separate compartment from the rest of the endoplasmic reticulum. Its lumen is continuous with that of the smooth ER and with the perinuclear space between the two membranes of the nuclear envelope, and the outer nuclear membrane is continuous with the ER membrane and also carries ribosomes. The RER is especially extensive in cells that secrete large amounts of protein, such as the pancreatic acinar cells, plasma cells (which secrete antibodies), fibroblasts, and hepatocytes, and it is less developed in cells that make few secreted proteins. In the light microscope, the abundant RER of such cells is seen as basophilic regions of the cytoplasm, which were called ergastoplasm or Nissl substance in neurons. The ER was first seen by Keith Porter, Albert Claude, and Ernest Fullam in the 1940s, and the ribosomes on the RER and the process of protein secretion were studied by George Palade, who shared the 1974 Nobel Prize in Physiology or Medicine with Albert Claude and Christian de Duve.

Membrane and lumen

The ER is the largest membrane system of most cells, and it can account for more than half of the membrane of the cell. The ER lumen is a distinct environment. It has a high concentration of calcium (about 0.1 to 1 mM, compared with about 100 nM in the cytosol) and an oxidizing redox potential that favors the formation of disulfide bonds, and it contains abundant chaperones and enzymes. The ER membrane is maintained in two shapes by different proteins. Flat sheets, which carry most of the ribosomes, are stabilized by proteins such as CLIMP-63 and by the ribosomes and translocons themselves. Tubules and the edges of sheets, which have a high membrane curvature, are shaped by reticulon and REEP proteins. Atlastin GTPases mediate the fusion of tubules and generate the three-way junctions of the network. The balance between sheets and tubules differs among cell types and changes with the demand for protein synthesis.

Ribosome attachment

Ribosomes bind to the RER through the translocon (also called the Sec61 complex) and through other membrane proteins. They are not permanently bound. The same ribosome that is attached to the RER during the synthesis of one protein returns to the cytosolic pool after translation ends, and it can synthesize a cytosolic protein in a later round. Polyribosomes (several ribosomes on one mRNA) are often attached to the RER at several points. The distinction between "free" and "bound" ribosomes is therefore a matter of what they are translating at the time.

Signal sequences and the signal recognition particle

Proteins that are destined for the secretory pathway have a signal peptide at the N-terminus, or an internal signal sequence or transmembrane segment. A typical signal peptide is 15 to 30 amino acids long, with a positively charged N-terminal region, a central hydrophobic core of 6 to 15 residues, and a polar C-terminal region that contains the site of cleavage. It is a "zip code" in an analogy, and the particular amino acid sequence is not conserved, whereas the hydrophobic character is.

In the cotranslational pathway, the signal peptide emerges from the ribosome and is bound by the signal recognition particle (SRP), a ribonucleoprotein complex that contains a 7SL RNA and six proteins (SRP54 binds the signal sequence). The binding of the SRP slows or arrests translation in many cases, which gives time for targeting and prevents the aggregation of hydrophobic sequences in the cytosol. The complex of ribosome, nascent chain, and SRP binds to the SRP receptor (a heterodimer of SRα and SRβ) on the ER membrane. GTP binding and hydrolysis by SRP and by its receptor control the steps of targeting and release. The ribosome is then transferred to the Sec61 translocon, translation resumes, and the SRP is released. The SRP is not a "tugboat" that pulls the ribosome. It binds the ribosome and the signal sequence, and the movement of the complex to the membrane is by diffusion and by the affinity for the receptor.

The Sec61 translocon

The Sec61 complex is a protein-conducting channel in the ER membrane. Its α subunit forms an hourglass-shaped pore with a lateral gate that opens to the lipid bilayer, and a plug that seals the channel when it is not in use. The ribosome docks on the cytosolic side so that the tunnel of the ribosome is aligned with the pore. A signal peptide opens the plug and inserts into the lateral gate. As translation continues, the polypeptide is threaded through the pore into the lumen. In the case of a membrane protein, a hydrophobic transmembrane segment exits laterally through the gate into the lipid, and the topology (the number of passes and the orientation) depends on the positive and negative charges next to the hydrophobic segments (the "positive-inside rule") and on other signals. Accessory complexes help in specific cases. The translocon-associated protein complex, the oligosaccharyltransferase, and the signal peptidase are found near the channel, and the EMC (ER membrane protein complex) and other insertases help with the insertion of membrane proteins that have few transmembrane segments.

The signal peptidase in the lumen cleaves the signal peptide from most soluble secretory proteins during or just after translocation. Some proteins use alternative routes. Tail-anchored proteins, which have a single transmembrane segment at the C-terminus, are inserted after translation by the GET (TRC40) pathway. Short proteins can be translocated after translation with the help of the Sec62 and Sec63 proteins and the lumenal chaperone BiP, and this is the main route in yeast. BiP also acts as a ratchet in post-translational translocation.

Lumenal chaperones and the ratchet

As the polypeptide enters the lumen, it is bound by the Hsp70 chaperone BiP (also called GRP78), which uses the energy of ATP hydrolysis and prevents the chain from sliding back. BiP is regulated by J-domain proteins and nucleotide exchange factors. Other chaperones include the Hsp90 homolog GRP94, the lectin chaperones calnexin and calreticulin, and protein disulfide isomerases.

Quantitative Comparison of Core Molecular Complexes of the Rough Endoplasmic Reticulum

Disulfide bonds and oxidative folding

The lumen is an oxidizing environment, with a ratio of reduced to oxidized glutathione that is much lower than that of the cytosol. Protein disulfide isomerase (PDI) catalyzes the formation, breaking, and rearrangement of disulfide bonds between cysteine residues, and the ER oxidoreductin Ero1 reoxidizes PDI, with oxygen as the final electron acceptor, which produces hydrogen peroxide. Disulfide bonds stabilize secreted and membrane proteins, which are in oxidizing environments outside the cell.

N-linked glycosylation

Most proteins that enter the ER are glycosylated. The oligosaccharyltransferase complex transfers a preassembled oligosaccharide of 14 sugar residues (Glc₃Man₉GlcNAc₂) from a lipid carrier, dolichol phosphate, to the asparagine in the sequence Asn-X-Ser or Asn-X-Thr (where X is any amino acid except proline) as the polypeptide emerges in the lumen. Glucosidases then trim the two outer glucose residues. A monoglucosylated glycan is recognized by the lectin chaperones calnexin and calreticulin, which retain the protein in the folding cycle and recruit ERp57, a PDI family protein. When the last glucose is removed by glucosidase II, the protein is released. If it is not yet folded, it is recognized by UDP-glucose:glycoprotein glucosyltransferase (UGGT), which adds a glucose back and sends it into another round. Folded proteins leave, and persistently unfolded proteins are marked for degradation by the trimming of mannose residues. The glycan therefore acts as a timer for folding and as a tag for quality control, in addition to its roles in stability and recognition. Further processing of the glycans is done in the Golgi apparatus, so that the "complex" carbohydrate chains are made there, and not in the RER.

Other modifications

Other modifications in the ER lumen include the addition of glycosylphosphatidylinositol (GPI) anchors to some proteins, the hydroxylation of proline and lysine residues in procollagen, the assembly of multi-subunit complexes such as antibodies and collagen triple helices, and the isomerization of proline by peptidyl-prolyl isomerases.

ER-associated degradation

Proteins that do not fold correctly are retained by chaperones and, if folding fails, they are removed by ER-associated degradation (ERAD). They are recognized, partly by lectins that bind trimmed glycans (for example EDEM and OS-9), and are retrotranslocated through a channel, which includes the E3 ubiquitin ligase Hrd1, to the cytosol. There they are ubiquitinated, extracted from the membrane by the AAA ATPase p97 (VCP), and degraded by the proteasome. This is a more complex process than "ejection and shredding". Some misfolded proteins are removed by autophagy of the ER (ER-phagy). Mutations that cause misfolding can lead to disease through loss of function of the protein, as in the case of the most common mutation of CFTR (ΔF508) in cystic fibrosis, in which the protein is degraded by ERAD and does not reach the cell surface, or through the toxic effects of accumulation of misfolded protein.

The unfolded protein response

When the load of unfolded proteins exceeds the capacity of the ER, the cell responds with the unfolded protein response (UPR), which has three branches, each with a sensor in the ER membrane:

• IRE1 is a kinase and an endoribonuclease. Its activation causes the unconventional splicing of the mRNA for XBP1 (in the cytosol), which makes an active transcription factor that increases the production of chaperones and of ERAD components, and expands the ER.
• PERK is a kinase that phosphorylates the translation initiation factor eIF2α, which decreases general protein synthesis and so reduces the load of the ER. It increases the translation of ATF4, which regulates genes for amino acid metabolism, antioxidant responses, and, when the stress persists, the pro-apoptotic factor CHOP.
• ATF6 is transported to the Golgi apparatus when stress is detected, where it is cleaved by proteases, and its cytosolic fragment goes to the nucleus and increases the expression of chaperones.

Under stress, BiP is titrated away from the sensors by unfolded proteins, which activates them. The UPR initially restores homeostasis, and if the stress is severe or prolonged, it signals apoptosis. ER stress is implicated in diabetes (in the stress of pancreatic β cells), in neurodegenerative diseases, in cancer, and in infections.

Export from the ER

Properly folded proteins leave the RER at ER exit sites (transitional ER), which are regions with few ribosomes. There, the COPII coat (the proteins Sar1, Sec23/24, and Sec13/31) assembles on the membrane and selects cargo, and it forms vesicles and tubules that carry the cargo to the ER-Golgi intermediate compartment (ERGIC) and to the cis-Golgi. Cargo receptors (such as the ERGIC-53 lectin) help to load some proteins. Proteins that belong in the ER and that escape are returned by the COPI coat. Soluble ER residents have a C-terminal KDEL sequence that is recognized by the KDEL receptor in the Golgi apparatus, which carries them back. The statement that vesicles are transported "directly" along the cytoskeleton is simplified, because the transport in many cells is by microtubule motors and tethering factors, and in some cases the vesicles fuse with others before reaching the Golgi. In many mammalian cells, the ER and the Golgi are close to each other, and the distance is short.

Smooth ER and contact sites

The smooth ER lacks ribosomes and is the site of the synthesis of lipids and steroids, the detoxification of drugs by cytochrome P450 enzymes, the metabolism of glycogen in the liver (glucose-6-phosphatase), and, in muscle, the storage and release of calcium (the sarcoplasmic reticulum). The smooth and rough ER are parts of the same network, with a continuous lumen, and the proportion of the two differs among cell types. The ER also forms contact sites with the plasma membrane, mitochondria, endosomes, lipid droplets, and the Golgi apparatus. At these sites, lipids and calcium are transferred without vesicles. The synthesis of most membrane phospholipids, and of cholesterol, also takes place in the ER, and so the RER is also a source of membrane for the secretory pathway. In addition, the ER is the largest store of calcium in the cell, and it takes part in calcium signaling through IP₃ receptors and ryanodine receptors, with the SERCA pump that loads calcium. The ER is also needed for the synthesis of lipid components of lipoproteins and for the formation of lipid droplets.

The rate of protein synthesis on the RER can be large. A pancreatic acinar cell or a plasma cell may secrete thousands of protein molecules per second. The mean rate of translation by a ribosome in eukaryotes is about 3 to 5 amino acids per second, so that a secretory protein of 400 residues needs about 80 to 130 seconds to be made. The oligosaccharyltransferase must be fast enough to match this. If the cell has N active ribosomes on the RER and a typical protein has L residues, the output of proteins is approximately:

where v is the elongation rate in residues per second. For example, for N = 10⁶ active ribosomes, v = 4 residues per second, and L = 400, the output is J ≈ 10⁶ × 4 / 400 = 10⁴ proteins per second. This is of the order of the highest rates of secretion by specialized cells. A fraction of cells' ribosomes is bound to the ER, and the numbers differ among cell types.

Defects in the function of the RER cause or contribute to several diseases. These include alpha-1 antitrypsin deficiency (the misfolded Z variant accumulates in the ER of hepatocytes, which causes liver disease, and its deficiency in the lung causes emphysema), osteogenesis imperfecta (mutations in collagen that disturb folding), cystic fibrosis, diseases of ER storage, and diseases of glycosylation. Mutations in the genes for the SRP pathway or the translocon are rare causes of disease, such as the Sec61 mutations that cause some forms of polycystic liver disease and of immunodeficiency. Some viruses (such as flaviviruses and coronaviruses) remodel the ER to form replication organelles, and some bacterial toxins (cholera toxin, Shiga toxin) use retrograde transport to reach the ER and then exit to the cytosol by the ERAD machinery. Inhibitors of the translocon, the proteasome, and p97 are used as research tools and in some cases as drugs, such as bortezomib in multiple myeloma, a cancer of plasma cells, which depend on the RER for the production of antibodies.

The RER is studied by electron microscopy, by fluorescence microscopy with ER markers (such as KDEL-tagged proteins and Sec61β), by biochemical fractionation, in which the ER is isolated as microsomes (vesicles that form when cells are broken), and by ribosome profiling and proximity labeling to identify mRNAs that are translated on the ER. Microsomes with and without ribosomes can be separated by density, and the in vitro translation of mRNA in the presence of microsomes was used to show the signal hypothesis of Günter Blobel, who received the 1999 Nobel Prize in Physiology or Medicine for it.

How does the Sec61 translocon insert transmembrane helices into the lipid bilayer without leaking luminal calcium?

During co-translational translocation, the ribosome-Sec61 junction and a ring of six hydrophobic isoleucine residues inside the Sec61α hourglass pore form a tight gasket around the nascent polypeptide chain, preventing Ca²⁺ ions from leaking out of the lumen. When a hydrophobic transmembrane segment (~18–25 non-polar residues) enters the pore, the Sec61α lateral gate between transmembrane helices TM2b and TM7 parts laterally, allowing the helix to partition thermodynamically into the phospholipid bilayer.

How does UDP-glucose:glycoprotein glucosyltransferase (UGGT) act as a folding sensor in the calnexin cycle?

After oligosaccharyltransferase attaches Glc₃Man₉GlcNAc₂ to a nascent polypeptide, glucosidases I and II trim two glucose residues to generate a monoglucosylated glycan that binds the lectin chaperones calnexin and calreticulin. Once glucosidase II cleaves the final glucose to release the substrate, UGGT inspects the polypeptide conformation; if exposed hydrophobic patches remain, UGGT selectively re-glucosylates the glycan to force another folding cycle.

Why does the ΔF508 mutation in CFTR cause cystic fibrosis even though the mutant channel retains partial chloride conductance?

Deletion of phenylalanine 508 (ΔF508) in the nucleotide-binding domain 1 (NBD1) of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) slows domain folding kinetics on the rough endoplasmic reticulum without completely abolishing channel gating. However, the stringent ER quality control machinery (Hsp70/CHIP and Hrd1 ERAD complexes) recognizes the delayed folding intermediate, polyubiquitinates ~99% of nascent ΔF508-CFTR polypeptides, and retrotranslocates them for proteasomal destruction before they can traffic to the apical plasma membrane.

• Endoplasmic reticulum
• Signal recognition particle
• Unfolded protein response
• Golgi apparatus
• Protein targeting
• Protein folding
• Ribosome