The Endoplasmic Reticulum and Protein Secretion

The role of the endoplasmic reticulum in protein processing and sorting was first demonstrated by George Palade and his colleagues in the 1960s (Figure 9.2). These investigators studied the fate of newly synthesized proteins in specialized cells of the pancreas (pancreatic acinar cells) that secrete digestive enzymes into the small intestine. Because most proteins synthesized by these cells are secreted, Palade and coworkers were able to study the pathway taken by secreted proteins simply by labeling newly synthesized proteins with radioactive amino acids. The location of the radiolabeled proteins within the cell was then determined by autoradiography, revealing the cellular sites involved in the events leading to protein secretion. After a brief exposure of pancreatic acinar cells to radioactive amino acids, newly synthesized proteins were detected in the rough ER, which was therefore identified as the site of synthesis of proteins destined for secretion. If the cells were then incubated for a short time in media containing nonradioactive amino acids (a process known as a chase), the radiolabeled proteins were detected in the Golgi apparatus. Following longer chase periods, the radiolabeled proteins traveled from the Golgi apparatus to the cell surface in secretory vesicles, which then fused with the plasma membrane to release their contents outside of the cell.

Figure 9.2

The secretory pathway. Pancreatic acinar cells, which secrete most of their newly synthesized proteins into the digestive tract, were labeled with radioactive amino acids to study the intracellular pathway taken by secreted proteins. After a short incubation (more...)

These experiments defined a pathway taken by secreted proteins, the secretory pathway: rough ER → Golgi → secretory vesicles → cell exterior. Further studies extended these results and demonstrated that this pathway is not restricted to proteins destined for secretion from the cell. Plasma membrane and lysosomal proteins also travel from the rough ER to the Golgi and then to their final destinations. Still other proteins travel through the initial steps of the secretory pathway but are then retained and function within either the ER or the Golgi apparatus.

The entrance of proteins into the ER thus represents a major branch point for the traffic of proteins within eukaryotic cells. Proteins destined for secretion or incorporation into the ER, Golgi apparatus, lysosomes, or plasma membrane are initially targeted to the ER. In mammalian cells, most proteins are transferred into the ER while they are being translated on membrane-bound ribosomes (Figure 9.3). In contrast, proteins destined to remain in the cytosol or to be incorporated into the nucleus, mitochondria, chloroplasts, or peroxisomes are synthesized on free ribosomes and released into the cytosol when their translation is complete.

Figure 9.3

Overview of protein sorting. In mammalian cells, the initial sorting of proteins to the ER takes place while translation is in progress. Proteins synthesized on free ribosomes either remain in the cytosol or are transported to the nucleus, mitochondria, (more...)

Targeting Proteins to the Endoplasmic Reticulum

Proteins can be translocated into the ER either during their synthesis on membrane-bound ribosomes (cotranslational translocation) or after their translation has been completed on free ribosomes in the cytosol (posttranslational translocation). In mammalian cells, most proteins enter the ER co-translationally, whereas both cotranslational and posttranslational pathways are used in yeast. The first step in the cotranslational pathway is the association of ribosomes with the ER. Ribosomes are targeted for binding to the ER membrane by the amino acid sequence of the polypeptide chain being synthesized, rather than by intrinsic properties of the ribosome itself. Free and membrane-bound ribosomes are functionally indistinguishable, and all protein synthesis initiates on ribosomes that are free in the cytosol. Ribosomes engaged in the synthesis of proteins that are destined for secretion are then targeted to the endoplasmic reticulum by a signal sequence at the amino terminus of the growing polypeptide chain. These signal sequences are short stretches of hydrophobic amino acids that are cleaved from the polypeptide chain during its transfer into the ER lumen.

The general role of signal sequences in targeting proteins to their appropriate locations within the cell was first elucidated by studies of the import of secretory proteins into the ER. These experiments used in vitro preparations of rough ER, which were isolated from cell extracts by density-gradient centrifugation (Figure 9.4). When cells are disrupted, the ER breaks up into small vesicles called microsomes. Because the vesicles derived from the rough ER are covered with ribosomes, they can be separated from similar vesicles derived from the smooth ER or from other membranes (e.g., the plasma membrane). In particular, the large amount of RNA within ribosomes increases the density of the membrane vesicles to which they are attached, allowing purification of vesicles derived from the rough ER (rough microsomes) by equilibrium centrifugation in density gradients.

Figure 9.4

Isolation of rough ER. When cells are disrupted, the ER fragments into small vesicles called microsomes. The microsomes derived from the rough ER (rough microsomes) are lined with ribosomes on their outer surface. Because ribosomes contain large amounts (more...)

David Sabatini and Günter Blobel first proposed in 1971 that the signal for ribosome attachment to the ER was an amino acid sequence near the amino terminus of the growing polypeptide chain. This hypothesis was supported by the results of in vitro translation of mRNAs encoding secreted proteins, such as immunoglobulins (Figure 9.5). If an mRNA encoding a secreted protein was translated on free ribosomes in vitro, it was found that the protein produced was slightly larger than the normal secreted protein. If microsomes were added to the system, however, the in vitro-translated protein was incorporated into the microsomes and cleaved to the correct size. These experiments led to a more detailed formulation of the signal hypothesis, which proposed that an amino-terminal leader sequence targets the polypeptide chain to the microsomes and is then cleaved by a microsomal protease. Many subsequent findings have substantiated this model, including recombinant DNA experiments demonstrating that addition of a signal sequence to a normally nonsecreted protein is sufficient to direct the incorporation of the recombinant protein into the rough ER.

Figure 9.5

Incorporation of secretory proteins into microsomes. Secretory proteins are targeted to the ER by a signal sequence at their amino (N) terminus, which is removed during incorporation of the growing polypeptide chain into the ER. This was demonstrated (more...)

The mechanism by which secretory proteins are targeted to the ER during their translation (the cotranslational pathway) is now well understood. The signal sequences span about 20 amino acids, including a stretch of hydrophobic residues, usually at the amino terminus of the polypeptide chain (Figure 9.6). As they emerge from the ribosome, signal sequences are recognized and bound by a signal recognition particle (SRP) consisting of six polypeptides and a small cytoplasmic RNA (7SL RNA). SRP binds the ribosome as well as the signal sequence, inhibiting further translation and targeting the entire complex (the SRP, ribosome, and growing polypeptide chain) to the rough ER by binding to the SRP receptor on the ER membrane (Figure 9.7). Binding to the receptor releases the SRP from both the ribosome and the signal sequence of the growing polypeptide chain. The ribosome then binds to a protein translocation complex in the ER membrane, and the signal sequence is inserted into a membrane channel. In both yeast and mammalian cells, the translocation channels through the ER membrane are complexes of three transmembrane proteins, called the Sec61 proteins. The yeast and mammalian Sec61 proteins are closely related to the plasma membrane proteins that translocate secreted polypeptides in bacteria, demonstrating a striking conservation of the protein secretion machinery in prokaryotic and eukaryotic cells. Transfer of the ribosome from the SRP to the Sec61 complex allows translation to resume, and the growing polypeptide chain is transferred directly into the Sec61 channel and across the ER membrane as translation proceeds. Thus, the process of protein synthesis directly drives the transfer of growing polypeptide chains through the Sec61 channel and into the ER. As translocation proceeds, the signal sequence is cleaved by signal peptidase and the polypeptide is released into the lumen of the ER.

Figure 9.6

The signal sequence of growth hormone. Most signal sequences contain a stretch of hydrophobic amino acids, preceded by basic residues (e.g., arginine).

Figure 9.7

Cotranslational targeting of secretory proteins to the ER. Step 1: As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP). Step 2: The SRP escorts the complex to the ER membrane, where it (more...)

Many proteins in yeast, as well as a few proteins in mammalian cells, are targeted to the ER after their translation is complete (posttranslational translocation), rather than being transferred into the ER during synthesis on membrane-bound ribosomes. These proteins are synthesized on free cytosolic ribosomes, and their posttranslational incorporation into the ER does not require SRP. Instead, their signal sequences are recognized by distinct receptor proteins (the Sec62/63 complex) associated with the Sec61 complex in the ER membrane (Figure 9.8). Cytosolic chaperones are required to maintain the polypeptide chains in an unfolded conformation so they can enter the Sec61 channel, and another chaperone within the ER (called BiP) is required to pull the polypeptide chain through the channel and into the ER. It appears that the binding of polypeptide chains to BiP is needed to drive the posttranslational translocation of proteins into the ER, whereas the cotranslational translocation of growing polypeptide chains is driven directly by the process of protein synthesis.

Figure 9.8

Posttranslational translocation of proteins into the ER. Proteins destined for posttranslational import to the ER are synthesized on free ribosomes and maintained in an unfolded conformation by cytosolic chaperones. Their signal sequences are recognized (more...)

Insertion of Proteins into the ER Membrane

Proteins destined for secretion or residence within the lumen of the ER, Golgi apparatus, or lysosomes are translocated across the ER membrane and released into the lumen of the ER as already described. However, proteins destined for incorporation into the plasma membrane or the membranes of the ER, Golgi, or lysosomes are initially inserted into the ER membrane instead of being released into the lumen. From the ER membrane, they proceed to their final destination along the same pathway as that of secretory proteins: ER→ Golgi→ plasma membrane or lysosomes. These proteins are transported along this pathway as membrane components, however, rather than as soluble proteins.

Integral membrane proteins are embedded in the membrane by hydrophobic regions that span the phospholipid bilayer (see Figure 2.48). The membrane-spanning portions of these proteins are usually α-helical regions consisting of 20 to 25 hydrophobic amino acids. The formation of an α helix maximizes hydrogen bonding between the peptide bonds, and the hydrophobic amino acid side chains interact with the fatty acid tails of the phospholipids. However, different integral membrane proteins differ in how they are inserted (Figure 9.9). For example, whereas some integral membrane proteins span the membrane only once, others have multiple membrane-spanning regions. In addition, some proteins are oriented in the membrane with their amino terminus on the cytosolic side; others have their carboxy terminus exposed to the cytosol. These orientations of proteins inserted into the ER, Golgi, lysosomal, and plasma membranes are established as the growing polypeptide chains are translocated into the ER. The lumen of the ER is topologically equivalent to the exterior of the cell, so the domains of plasma membrane proteins that are exposed on the cell surface correspond to the regions of polypeptide chains that are translocated into the ER (Figure 9.10).

Figure 9.9

Orientations of membrane proteins. Integral membrane proteins span the membrane via α-helical regions of 20 to 25 hydrophobic amino acids, which can be inserted in a variety of orientations. The two proteins at left and center each span the membrane (more...)

Figure 9.10

Topology of the secretory pathway. The lumens of the endoplasmic reticulum and Golgi apparatus are topologically equivalent to the exterior of the cell. Consequently, those portions of polypeptide chains that are translocated into the ER are exposed on (more...)

The most straightforward mode of insertion into the ER membrane results in the synthesis of transmembrane proteins oriented with their carboxy termini exposed to the cytosol (Figure 9.11). These proteins have a normal amino-terminal signal sequence, which is cleaved by signal peptidase during translocation of the polypeptide chain across the ER membrane through the Sec61 channel. They are then anchored in the membrane by a second membrane-spanning α helix in the middle of the protein. This transmembrane sequence, called a stop-transfer sequence, signals closure of the Sec61 channel. Further translocation of the polypeptide chain across the ER membrane is thus blocked, so the carboxy-terminal portion of the growing polypeptide chain is synthesized in the cytosol. The transmembrane domain then exits the translocation channel laterally to enter the lipid bilayer. The insertion of these proteins in the membrane thus involves the sequential action of two distinct elements: a cleavable amino-terminal signal sequence that initiates translocation across the membrane and a transmembrane stop-transfer sequence that anchors the protein in the membrane.

Figure 9.11

Insertion of a membrane protein with a cleavable signal sequence and a single stop-transfer sequence. The signal sequence is cleaved as the polypeptide chain crosses the membrane, so the amino terminus of the polypeptide chain is exposed in the ER lumen. (more...)

Proteins can also be anchored in the ER membrane by internal signal sequences that are not cleaved by signal peptidase (Figure 9.12). These internal signal sequences are recognized by the SRP and brought to the ER membrane as already discussed. Because they are not cleaved by signal peptidase, however, these signal sequences act as transmembrane α helices that exit the translocation channel and anchor proteins in the ER membrane. Importantly, internal signal sequences can be oriented so as to direct the translocation of either the amino or carboxy terminus of the polypeptide chain across the membrane. Therefore, depending on the orientation of the signal sequence, proteins inserted into the membrane by this mechanism can have either their amino or carboxy terminus exposed to the cytosol.

Figure 9.12

Insertion of membrane proteins with internal noncleavable signal sequences. Internal noncleavable signal sequences can lead to the insertion of polypeptide chains in either orientation in the ER membrane. (A) The signal sequence directs insertion of the (more...)

Proteins that span the membrane multiple times are thought to be inserted as a result of an alternating series of internal signal sequences and transmembrane stop-transfer sequences. For example, an internal signal sequence can result in membrane insertion of a polypeptide chain with its amino terminus on the cytosolic side (Figure 9.13). If a stop-transfer sequence is then encountered, the polypeptide will form a loop in the ER lumen, and protein synthesis will continue on the cytosolic side of the membrane. If a second signal sequence is encountered, the growing polypeptide chain will again be inserted into the ER, forming another looped domain on the cytosolic side of the membrane. This can be followed by yet another stop-transfer sequence and so forth, so that an alternating series of signal and stop-transfer sequences can result in the insertion of proteins that span the membrane multiple times, with looped domains exposed on both the lumenal and cytosolic sides.

Figure 9.13

Insertion of a protein that spans the membrane multiple times. In this example, an internal signal sequence results in insertion of the polypeptide chain with its amino terminus on the cytosolic side of the membrane. A stop-transfer sequence then signals (more...)

Protein Folding and Processing in the ER

The folding of polypeptide chains into their correct three-dimensional conformations, the assembly of polypeptides into multisubunit proteins, and the covalent modifications involved in protein processing were discussed in Chapter 7. For proteins that enter the secretory pathway, many of these events occur either during translocation across the ER membrane or within the ER lumen. One such processing event is the proteolytic cleavage of the signal peptide as the polypeptide chain is translocated across the ER membrane. The ER is also the site of protein folding, assembly of multisubunit proteins, disulfide bond formation, the initial stages of glycosylation, and the addition of glycolipid anchors to some plasma membrane proteins. Indeed, the primary role of lumenal ER proteins is to catalyze the folding and assembly of newly translocated polypeptides.

As already discussed, proteins are translocated across the ER membrane as unfolded polypeptide chains while their translation is still in progress. These polypeptides, therefore, fold into their three-dimensional conformations within the ER, assisted by the molecular chaperones that facilitate the folding of polypeptide chains (see Chapter 7). For example, one of the major proteins within the ER lumen is a member of the Hsp70 family of chaperones called BiP. BiP is thought to bind to the unfolded polypeptide chain as it crosses the membrane and then mediates protein folding and the assembly of multisubunit proteins within the ER (Figure 9.14). Correctly assembled proteins are released from BiP and are available for transport to the Golgi apparatus. Abnormally folded or improperly assembled proteins, however, remain bound to BiP and are consequently retained within the ER or degraded, rather than being transported farther along the secretory pathway.

Figure 9.14

Protein folding in the ER. The molecular chaperone BiP binds to polypeptide chains as they cross the ER membrane and facilitates protein folding and assembly within the ER.

The formation of disulfide bonds between the side chains of cysteine residues is an important aspect of protein folding and assembly within the ER. These bonds do not form in the cytosol, which is characterized by a reducing environment that maintains cysteine residues in their reduced (—SH) state. In the ER, however, an oxidizing environment promotes disulfide (S—S) bond formation, and disulfide bonds formed in the ER play important roles in the structure of secreted and cell surface proteins. Disulfide bond formation is facilitated by the enzyme protein disulfide isomerase (see Figure 7.21), which is located in the ER lumen.

Proteins are also glycosylated on specific asparagine residues (N-linked glycosylation) within the ER while their translation is still in process (Figure 9.15). As discussed in Chapter 7, oligosaccharide units consisting of 14 sugar residues are added to acceptor asparagine residues of growing polypeptide chains as they are translocated into the ER. The oligosaccharide is synthesized on a lipid (dolichol) carrier anchored in the ER membrane. It is then transferred as a unit to acceptor asparagine residues in the consensus sequence Asn-X-Ser/Thr by a membrane-bound enzyme called oligosaccharyl transferase. Four sugar residues (three glucose and one mannose) are removed while the protein is still within the ER, and the protein is modified further after being transported to the Golgi apparatus (discussed later in this chapter).

Figure 9.15

Protein glycosylation in the ER.

Some proteins are anchored in the plasma membrane by glycolipids rather than by membrane-spanning regions of the polypeptide chain. Because these membrane-anchoring glycolipids contain phosphatidylinositol, they are called glycosylphosphatidylinositol (GPI) anchors, the structure of which was illustrated in Figure 7.32. The GPI anchors are assembled in the ER membrane. They are then added immediately after completion of protein synthesis to the carboxy terminus of some proteins anchored in the membrane by a C-terminal membrane-spanning region (Figure 9.16). The transmembrane region of the protein is exchanged for the GPI anchor, so these proteins remain attached to the membrane only by their associated glycolipid. Like transmembrane proteins, they are transported to the cell surface as membrane components via the secretory pathway. Their orientation within the ER dictates that GPI-anchored proteins are exposed on the outside of the cell, with the GPI anchor mediating their attachment to the plasma membrane.

Figure 9.16

Addition of GPI anchors. Glycosylphosphatidylinositol (GPI) anchors contain two fatty acid chains, an oligosaccharide portion consisting of inositol and other sugars, and ethanolamine (see Figure 7.32 for a more detailed structure). The GPI anchors are (more...)

The Smooth ER and Lipid Synthesis

In addition to its activities in the processing of secreted and membrane proteins, the ER is the major site at which membrane lipids are synthesized in eukaryotic cells. Because they are extremely hydrophobic, lipids are synthesized in association with already existing cellular membranes rather than in the aqueous environment of the cytosol. Although some lipids are synthesized in association with other membranes, most are synthesized in the ER. They are then transported from the ER to their ultimate destinations either in vesicles or by carrier proteins, as discussed later in this chapter and in Chapter 10.

The membranes of eukaryotic cells are composed of three main types of lipids: phospholipids, glycolipids, and cholesterol. Most of the phospholipids, which are the basic structural components of the membrane, are derived from glycerol. They are synthesized on the cytosolic side of the ER membrane, from water-soluble cytosolic precursors (Figure 9.17). Fatty acids are first transferred from coenzyme A carriers to glycerol-3-phosphate by a membrane-bound enzyme, and the resulting phospholipid (phosphatidic acid) is inserted into the membrane. Enzymes on the cytosolic face of the ER membrane then catalyze the addition of different polar head groups, resulting in formation of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, or phosphatidylinositol.

Figure 9.17

Synthesis of phospholipids. Glycerol phospholipids are synthesized in the ER membrane from cytosolic precursors. Two fatty acids linked to coenzyme A (CoA) carriers are first joined to glycerol-3-phosphate, yielding phosphatidic acid, which is simultaneously (more...)

The synthesis of these phospholipids on the cytosolic side of the ER membrane allows the hydrophobic fatty acid chains to remain buried in the membrane while membrane-bound enzymes catalyze their reactions with water-soluble precursors (e.g., CDP-choline) in the cytosol. Because of this topography, however, new phospholipids are added only to the cytosolic half of the ER membrane (Figure 9.18). To maintain a stable membrane, some of these newly synthesized phospholipids must therefore be transferred to the other (lumenal) half of the ER bilayer. This transfer does not occur spontaneously because it requires the passage of a polar head group through the membrane. Instead, membrane proteins called flippases catalyze the rapid translocation of phospholipids across the ER membrane, resulting in even growth of both halves of the bilayer.

Figure 9.18

Translocation of phospholipids across the ER membrane. Because phospholipids are synthesized on the cytosolic side of the ER membrane, they are added only to the cytosolic half of the bilayer. They are then translocated across the membrane by phospholipid (more...)

In addition to its role in synthesis of the glycerol phospholipids, the ER also serves as the major site of synthesis of two other membrane lipids: cholesterol and ceramide (Figure 9.19). As discussed later, ceramide is converted to either glycolipids or sphingomyelin (the only membrane phospholipid not derived from glycerol) in the Golgi apparatus. The ER is thus responsible for synthesis of either the final products or the precursors of all the major lipids of eukaryotic membranes.

Figure 9.19

Structure of cholesterol and ceramide. The hydrogens attached to the ring carbons of cholesterol are not shown.

Smooth ER is abundant in cell types that are particularly active in lipid metabolism. For example, steroid hormones are synthesized (from cholesterol) in the ER, so large amounts of smooth ER are found in steroid-producing cells, such as those in the testis and ovary. In addition, smooth ER is abundant in the liver, where it contains enzymes that metabolize various lipid-soluble compounds. These detoxifying enzymes inactivate a number of potentially harmful drugs (e.g., phenobarbital) by converting them to water-soluble compounds that can be eliminated from the body in the urine. The smooth ER is thus involved in multiple aspects of the metabolism of lipids and lipid-soluble compounds.

Export of Proteins and Lipids from the ER

Both proteins and lipids travel along the secretory pathway in transport vesicles, which bud from the membrane of one organelle and then fuse with the membrane of another. Thus, molecules are exported from the ER in vesicles that bud from the ER and carry their cargo first to the ER-Golgi intermediate compartment and then to the Golgi apparatus (Figure 9.20). Subsequent steps in the secretory pathway involve vesicular transport between different compartments of the Golgi and from the Golgi to lysosomes or the plasma membrane. In each case, proteins within the lumen of one organelle are packaged into the budding transport vesicle and then released into the lumen of the recipient organelle following vesicle fusion. Membrane proteins and lipids are transported similarly, and it is noteworthy that their topological orientation is maintained as they travel from one membrane-enclosed organelle to another. For example, the domains of a protein exposed on the cytosolic side of the ER membrane will also be exposed on the cytosolic side of the Golgi and plasma membranes, whereas protein domains exposed on the lumenal side of the ER membrane will be exposed on the lumenal side of the Golgi and on the exterior of the cell (see Figure 9.10).

Figure 9.20

Vesicular transport from the ER to the Golgi. Proteins and lipids are carried from the ER to the Golgi in transport vesicles that bud from the membrane of the ER and then fuse to form the vesicles and tubules of the ER-Golgi intermediate compartment (ERGIC). (more...)

While most proteins travel from the ER to the Golgi, some proteins must be retained within the ER rather than proceeding along the secretory pathway. In particular, proteins that function within the ER (including BiP, signal peptidase, protein disulfide isomerase, and other enzymes discussed earlier) must be retained within that organelle. Export to the Golgi versus retention in the ER is thus the first branch point encountered by proteins being sorted to their correct destinations in the secretory pathway. Similar branch points arise at each subsequent stage of transport, such as retention in the Golgi versus export to lysosomes or the plasma membrane. In each case, specific localization signals target proteins to their correct intracellular destinations.

The distinction between proteins exported from and those retained in the ER appears to be governed by two distinct types of targeting sequences that specifically mark proteins as either (1) destined for transport to the Golgi or (2) destined for retention in the ER. Many proteins are retained in the ER lumen as a result of the presence of the targeting sequence Lys-Asp-Glu-Leu (KDEL, in the single-letter code) at their carboxy terminus. If this sequence is deleted from a protein that is normally retained in the ER (e.g., BiP), the mutated protein is instead transported to the Golgi and secreted from the cell. Conversely, addition of the KDEL sequence to the carboxy terminus of proteins that are normally secreted causes them to be retained in the ER. The retention of some transmembrane proteins in the ER is similarly dictated by short C-terminal sequences that contain two lysine residues (KKXX sequences).

Interestingly, the KDEL and KKXX signals do not prevent soluble ER proteins from being packaged into vesicles and carried to the Golgi. Instead, these signals cause resident ER proteins to be selectively retrieved from the ER-Golgi intermediate compartment or the Golgi complex and returned to the ER via a recycling pathway (Figure 9.21). Proteins bearing the KDEL and KKXX sequences appear to bind to specific recycling receptors in the membranes of these compartments and are then selectively transported back to the ER.

Figure 9.21

Retrieval of resident ER proteins. Proteins destined to remain in the lumen of the ER are marked by the sequence Lys-Asp-Glu-Leu (KDEL) at their carboxy terminus. These proteins are exported from the ER to the Golgi in the nonselective bulk flow of proteins (more...)

The action of the KDEL and KKXX sequences as retention/retrieval signals indicates that there is a nonselective bulk flow of proteins through the secretory pathway leading from the ER to the cell surface. This bulk flow from the ER to the Golgi may be responsible for the export of many proteins from the ER. However, it also appears that some proteins destined for secretion are marked by signals that actively direct their export from the ER. Protein export from the ER can thus take place not only by bulk flow, but also by a regulated pathway that specifically recognizes targeting signals that mediate selective transport of proteins to the Golgi apparatus.

Box

Key Experiment: The Signal Hypothesis.