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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 17.6Post-Translational Modifications and Quality Control in the Rough ER

Newly synthesized polypeptides in the membrane and lumen of the ER undergo five principal modifications before they reach their final destinations:


Formation of disulfide bonds


Proper folding


Addition and processing of carbohydrates


Specific proteolytic cleavages


Assembly into multimeric proteins

The first two and the last of these modifications, which take place exclusively in the rough ER, are discussed in this section. Although addition of some carbohydrates and some proteolytic cleavages also occur in this organelle, many such modifications take place in the Golgi complex or forming secretory vesicles; we discuss these in later sections.

Only properly folded and assembled proteins are transported from the rough ER to the Golgi complex and ultimately to the cell surface or other final destination. Unfolded, misfolded, or partly folded and assembled proteins are selectively retained in the rough ER, or are retrieved from the cis-Golgi network and returned to the ER. Misfolded proteins and unassembled subunits of multimeric proteins often move from the ER lumen back through the translocon into the cytosol where they are degraded in proteasomes. We consider several examples of such “quality control” in the second half of this section.

Disulfide Bonds Are Formed and Rearranged in the ER Lumen

In Chapter 3 we learned that both intramolecular and intermolecular disulfide bonds (–S–S–) help stabilize the tertiary and quaternary structure of many proteins. These covalent bonds form by the oxidative linkage of sulfhydryl groups (–SH), also known as thiol groups, on two cysteine residues in the same or different polypeptide chains (see page 53). This reaction can proceed spontaneously only when sufficient oxidant is present. In eukaryotic cells, disulfide bonds are formed in the lumen of the rough ER but not in the cytosol. Thus disulfide bonds are found only in secretory proteins and in the exoplasmic domains of membrane proteins synthesized on the rough ER; soluble cytosolic proteins, synthesized on free ribosomes, lack disulfide bonds and depend on other interactions to stabilize their structures.

Localization of disulfide-bond formation in cells to the ER lumen indicates that this organelle has a favorable redox environment for oxidation of –SH groups, whereas the cytosol does not. The intracellular oxidant required for this reaction has not been identified. However, a mutation in one ER membrane protein renders yeast cells unable to generate disulfide bonds, suggesting that this protein may participate in oxidation of –SH groups in the ER lumen.

The tripeptide glutathione, often abbreviated G,

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is the major thiol-containing molecule in eukaryotic cells, and serves to prevent the formation of disulfide bonds in the cytosol. Glutathione shuttles between the reduced form (GSH) and the oxidized form, a disulfide-linked dimer (GSSG). The GSH:GSSG ratio is over 50:1 in the cytosol; oxidized GSSG in the cytosol is reduced by the enzyme glutathione reductase, using electrons from the potent reducing agent NADPH (see Figure 16-4):

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Thus cytosolic proteins in bacterial and eukaryotic cells do not utilize the disulfide bond as a stabilizing force because the high GSH:GSSG ratio would drive the system in the direction of Cys–SH and away from Cys–S–S–Cys.

In proteins that contain more than one disulfide bond, the proper pairing of cysteine residues is essential for normal structure and activity. Disulfide bonds sometimes are formed sequentially while a polypeptide is still growing on the ribosome. For instance, during synthesis of the immunoglobulin (Ig) light chain, which contains two disulfide bonds, the first and second cysteines closest to the N-terminus form a disulfide bond before the third cysteine has even been added to the nascent chain, automatically ensuring the correct pairing of cysteines. Similarly, the third cysteine pairs with the fourth to create the second disulfide bond.

The disulfide bonds in some proteins, however, do not link cysteines that occur sequentially in the amino acid sequence. For example, proinsulin has three disulfide bonds that link cysteines 1 and 4, 2 and 6, and 3 and 5. In this case, the first disulfide bonds that form spontaneously by oxidation of –SH groups may have to undergo rearrangement for the protein to achieve its proper folded conformation. In cells, the rearrangement of disulfide bonds is accelerated by the enzyme protein disulfide isomerase (PDI), which is found in abundance in the ER of secretory tissues in such organs as the liver and pancreas. In catalyzing rearrangement, PDI forms a disulfide-bonded substrate-enzyme intermediate (Figure 17-26a). This enzyme acts on a broad range of protein substrates, allowing them to reach their thermodynamically most stable conformations. Disulfide bonds generally form in a specific order, first stabilizing small domains of a polypeptide, then stabilizing the interactions of more distant segments; this phenomenon is illustrated by the folding of influenza HA protein discussed below. Although PDI occasionally binds to a protein that is irreversibly misfolded, it can escape from this useless intermediate as shown in Figure 17-26b.

Figure 17-26. (a) Rearrangement of disulfide bonds by protein disulfide isomerase (PDI).

Figure 17-26

(a) Rearrangement of disulfide bonds by protein disulfide isomerase (PDI). PDI contains an active-site with two reduced cysteine sulfhydryl (–SH) groups. The ionized (–S) form of one of these groups reacts with disulfide (S – S) (more...)

Image biotech.jpgMost proteins used for therapeutic purposes in humans or animals are secretory proteins stabilized by disulfide bonds. When recombinant DNA techniques are used to synthesize mammalian secretory proteins in bacterial cells, the proteins generally are not secreted (even when a bacterial signal sequence replaces the normal one). Rather, they accumulate in the cytosol where they often denature and precipitate, in part because disulfide bonds do not form. Sophisticated chemical methods are required to refold such bacterially produced proteins, an expensive process. Once it became clear that disulfide-bond formation occurs spontaneously only in the ER lumen, biotechnologists realized that bacterial cells are not suitable hosts for the synthesis of mammalian proteins that are normally stabilized by disulfide bonds. Nowadays, cultured animal cells generally are preferred for large-scale production of therapeutic proteins such as monoclonal antibodies, tissue plasminogen activator (an anticlotting agent), and erythropoietin, a hormone that stimulates production of red blood cells.

Correct Folding of Newly Made Proteins Is Facilitated by Several ER Proteins

As discussed in Chapter 3, many reduced, denatured proteins can spontaneously refold into their native state in vitro (see Figure 3-13). In most cases such refolding requires hours to reach completion, yet new secretory proteins generally fold into their proper conformation in the ER lumen within minutes after their synthesis. The ER contains several proteins that accelerate the folding of newly synthesized proteins within the ER lumen. Protein disulfide isomerase (PDI) is one such folding catalyst; the chaperone Hsc70 is another (see Figure 17-16). Like cytosolic Hsc70, this ER chaperone transiently binds to proteins and prevents them from misfolding or forming aggregates, thereby enhancing their ability to fold into the proper conformation. Two other ER proteins, the homologous lectins calnexin and calreticulin, bind to certain carbohydrates (discussed later) attached to newly made proteins and aid in protein folding (Figure 17-27).

Figure 17-27. Folding of the hemagglutinin (HA) precursor polypeptide HA0 and formation of an HA0 trimer within the ER.

Figure 17-27

Folding of the hemagglutinin (HA) precursor polypeptide HA0 and formation of an HA0 trimer within the ER. While the nascent chain is still growing, two protein-folding catalysts, calnexin and calreticulin, associate with it, and three disulfide bonds (more...)

Other important protein-folding catalysts are peptidyl-prolyl isomerases, a family of enzymes that accelerate the rotation about peptidyl-prolyl bonds in unfolded segments of a polypeptide:

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Such isomerizations frequently are the rate-limiting step in the folding of protein domains. Many peptidyl-prolyl isomerases can catalyze the rotation of any exposed peptidyl-prolyl bond, but some have very specific substrates. For example, in Drosophila an ER peptidyl-prolyl isomerase called NinaA is required for the folding of opsin, the membrane protein that absorbs light and triggers the visual response (see Figure 21-47). Flies with mutations in NinaA lack proper visual responses but otherwise are normal. This finding illustrates the remarkable specificity of some folding catalysts.

Assembly of Subunits into Multimeric Proteins Occurs in the ER

Many important secretory and membrane proteins are built of two or more polypeptides (or subunits). In all cases, these multimeric proteins are assembled in the ER. The number and relative positions of the subunits in a multimeric protein constitutes its quaternary structure. One important example is provided by the immunoglobulins, which contain two heavy (H) and two light (L) chains, all linked by S–S bonds (see Figure 3-21). Hemagglutinin (HA), the trimeric protein that forms the spikes protruding from the surface of the influenza virus particle (see Figure 6-13), provides another good illustration of protein folding and subunit assembly in the ER. Each spike is formed within the ER of an infected host cell from three copies of a precursor protein termed HA0, which has a single membrane-spanning α helix. In the Golgi complex, each of the three HA0 proteins is cleaved to form two polypeptides, HA1 and HA2; thus each spike in the virus particle contains three copies of HA1 and three of HA2 (see Figure 3-4). The trimer is stabilized by interactions between the exoplasmic domains of the constituent polypeptides as well as by interactions between the three cytosolic and membrane-spanning domains.

Each newly made HA0 polypeptide requires approximately 7 minutes to fold and be incorporated into a trimer. Several approaches have been used to detect different intermediates during the folding of HA0 monomers and their subsequent assembly into trimers in virus-infected cells (see Figure 17-27). One approach employs two monoclonal antibodies: one specific for HA0 monomers and one specific for the correctly folded trimer. In a typical experiment, virus-infected cells are pulse-labeled with a radioactive amino acid; at various times during the subsequent chase period, membranes are solubilized by detergent and exposed to the monomer- or trimer-specific antibody to precipitate HA0 protein. Immediately after the pulse, all of the radioactive HA0 protein is immunoprecipitated by the monomer-specific antibody. During the chase period, increasing proportions of the total radioactive HA0 protein react with the trimer-specific monoclonal antibody. Thus the time course of the monomer-to-trimer conversion within living cells can be followed, and the pattern of disulfide bonds in the immunoprecipitated monomers can be determined to identify various intermediates.

Only Properly Folded Proteins Are Transported from the Rough ER to the Golgi Complex

Almost any mutation in a secretory or membrane protein that prevents it from folding properly also blocks movement of the polypeptide from the lumen or membrane of the rough ER to the Golgi complex. In most cases, improperly folded proteins are permanently bound to the ER chaperones Hsc70 or calnexin. Thus these luminal folding catalysts perform two related functions: assisting in the folding of normal proteins by preventing their aggregation and binding to irreversibly misfolded proteins.

Image med.jpgOne medically important example is a mutation in the secretory protein α1-antiprotease, which is secreted by hepatocytes and macrophages; this protein binds to and inhibits trypsin and also the blood protease elastase. In the absence of α1-antiprotease, elastase degrades the fine tissue in the lung that participates in the absorption of oxygen. A genetic inability to produce functional α1-antiprotease, widespread in Caucasians, is the major genetic cause of emphysema (destruction of lung tissue by unchecked elastase). The defect is due to a single mutation in α1-antiprotease that causes lysine to replace glutamate 342. Although the mutant α1-antiprotease is synthesized in the rough ER, it does not fold properly, forming an almost crystalline aggregate that is not exported from the ER. In hepatocytes, the secretion of other proteins also becomes impaired as the rough ER is filled with aggregated α1-antiprotease.

Both mammalian cells and yeasts respond to the presence of unfolded proteins in the rough ER by increasing transcription of several genes encoding ER chaperones and other folding catalysts, including Hsc70, peptidyl-prolyl isomerase, and protein disulfide isomerase. A key element in this unfolded-protein response is IRE1, a transmembrane protein in the inner nuclear membrane, which is continuous with the ER membrane. As shown in Figure 17-28, binding of unfolded proteins in the ER lumen to the luminal surface of IRE1 promotes formation of the functional mRNA encoding transcription factor HAC1, which activates transcription of the genes induced in the unfolded-protein response. Thus, the increase in the level of HAC1 in response to an accumulation of unfolded proteins in the ER lumen leads to increased synthesis of precisely those proteins that assist in protein folding.

Figure 17-28. The unfolded-protein response.

Figure 17-28

The unfolded-protein response. IRE1 is a transmembrane protein in the inner nuclear membrane, a membrane that is continuous with the ER membrane. This multifunctional protein (green) has a binding site for unfolded proteins (blue) on its luminal surface; (more...)

Many Unassembled or Misfolded Proteins in the ER Are Transported to the Cytosol and Degraded

Mutant misfolded secretory and membrane proteins, as well as the unassembled subunits of multimeric proteins, often are degraded within an hour or two after their synthesis in the rough ER. For many years, researchers thought that proteolytic enzymes within the ER catalyzed degradation of misfolded or unassembled polypeptides, but such proteases were never found. Recent studies have shown that misfolded membrane and secretory proteins are transported from the ER lumen, “backwards” through the translocon, into the cytosol where they are degraded by the ubiquitin-mediated proteolytic pathway (see Figure 3-18). Two ubiquitin-conjugating enzymes form a complex localized to the cytosolic face of the ER and recruit a proteasome to the ER membrane where it can degrade misfolded proteins as they are extruded back through the translocon. The addition of ubiquitin to misfolded ER proteins, which is coupled to hydrolysis of ATP, may provide some of the energy required to drag these proteins back to the cytosol.

How misfolded membrane proteins are recognized and targeted to the translocon for export to the cytosol is not known. However, studies with the T-cell receptor suggest that the membrane-spanning region is critical to recognition. The α subunit of the T-cell receptor has a single membrane-spanning segment with two charged amino acids that are normally bound by ionic linkage (within the phospholipid bilayer) to residues of opposite charge in two other subunits of the T-cell receptor. If these other subunits are absent, the charged residues in the α subunit cause the entire polypeptide to assume an abnormal conformation, triggering its export via the translocon into the cytosol. Mutation of the charged residues to neutral ones prevents degradation of the α subunit when the other T-cell receptor subunits are absent — evidence that the membrane-spanning segment is recognized by the machinery that targets a protein back to the cytosol for degradation.

ER-Resident Proteins Often Are Retrieved from the Cis-Golgi

Another aspect of ER quality control is the retention in the ER lumen of soluble “resident” proteins such as Hsc70 and PDI, which catalyze the folding of newly made proteins. Most soluble proteins synthesized on the rough ER eventually are secreted from the cell surface or transported to the lumen of lysosomes (see Figure 17-13). How, then, are resident proteins retained in the ER lumen to carry out their work?

The answer lies in a specific C-terminal sequence present in resident ER proteins and in a receptor that recognizes this sequence. PDI, luminal Hsc70, and many other ER-resident proteins have a Lys-Asp-Glu-Leu (KDEL in the one-letter code) sequence at their C-terminus. Several experiments demonstrated that this KDEL sequence is both necessary and sufficient for retention in the ER. For instance, when a mutant PDI protein lacking these four residues is synthesized in a fibroblast, the protein is secreted. Moreover, if a protein that normally is secreted is altered so that it contains these four amino acids at its C-terminus, the protein is retained in the ER.

The KDEL receptor acts mainly to retrieve proteins with the KDEL recognition sequence that have escaped to the cis-Golgi network and return them to the ER. In support of this concept is the finding that most KDEL receptors are localized to the membranes of small transport vesicles shuttling between the ER and the cis-Golgi and to the membranes of the cis-Golgi reticulum (Figure 17-29). In addition, ER-localized proteins that carry the KDEL recognition sequence have oligosaccharide chains with modifications that are made by enzymes found only in the cis-Golgi or cis-Golgi reticulum; thus at some time these proteins must have been transported at least to the cis-Golgi network. Unfortunately, we do not yet know how the movements of the KDEL receptor are controlled.

Figure 17-29. Role of the KDEL receptor in the retrieval of ER-resident proteins.

Figure 17-29

Role of the KDEL receptor in the retrieval of ER-resident proteins. Many resident proteins in the ER lumen bear a C-terminal KDEL (Lys-Asp-Glu-Leu) sequence that localizes them to the ER. The KDEL receptor is located mainly in the cis-Golgi network and (more...)

Clearly, the transport of newly made proteins from the rough ER to the Golgi cisternae is a highly selective and regulated process; the selective entry of proteins into membrane-bounded transport vesicles is an important feature of protein targeting — one we will encounter several times in our study of the subsequent stages in the maturation of secretory and membrane proteins.


  •  The redox environment in the lumen of the rough ER is favorable for oxidation of cysteine sulfhydryl groups (–SH) to disulfide bonds (–S–S–), whereas this reaction does not occur in the cytosol. Thus disulfide bonds are common in secretory proteins and exoplasmic domains of membrane proteins, but are absent from soluble cytosolic proteins.
  •  Protein disulfide isomerase (PDI), an enzyme localized to the ER lumen, catalyzes the rearrangement of disulfide bonds, accelerating the folding of newly synthesized secretory and membrane proteins in the ER.
  •  The folding of many newly made proteins within the ER is facilitated by other folding catalysts such as peptidyl-prolyl isomerases and the lectins calnexin and calreticulin (see Figure 17-27).
  •  Assembly of subunits to form multimeric secretory and membrane proteins occurs in the ER.
  •  Only properly folded proteins are transported from the rough ER to the Golgi complex of vesicles.
  •  Abnormally folded proteins and unassembled subunits are selectively retained in the ER, either because they form aggregates or because they are permanently bound to Hsc70 or other ER chaperones.
  •  Unassembled or misfolded proteins in the ER often are transported back through the translocon to the cytosol where they are degraded in the ubiquitin/ proteasome pathway.
  •  Accumulation of misfolded proteins in the ER lumen induces increased production of ER protein-folding catalysts via the unfolded-protein response (see Figure17-28).
  •  Certain resident ER proteins are retained in the ER by a C-terminal KDEL sequence or are retrieved there from the cis-Golgi network by the KDEL receptor (see Figure 17-29).
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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21741