Medical Physiology, 3rd Edition

Synthesis and Recycling of Membrane Proteins

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 (see pp. 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 (see pp. 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 the rough ER (see pp. 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 (see pp. 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 of secretory proteins. It is now appreciated that the mRNA associated with the ER also encodes the cell's entire repertoire of membrane 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 not secretory proteins but rather the soluble cytosolic 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, some localization 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.

The nature of this signal was discovered in 1972 during studies of the biosynthesis of immunoglobulin light chains. Light chains synthesized in vitro, in the absence of rough ER membranes, have a 15–amino-acid extension at their amino terminus that is absent from the same light chains synthesized and secreted in vivo by B lymphocytes. Similar amino-terminal extensions are present on most secretory or membrane proteins but never on the soluble proteins of the cytosol. Although they vary in length and composition, these extensions are usually α-helical and are composed of strings of hydrophobic acids that are often preceded by short stretches of basic residues. These signal sequences, as they have come to be known, serve as the localization devices discussed above. As it emerges from a ribosome and is freely floating in the cytosol, the signal sequence of a nascent protein (Fig. 2-15, stage 1) targets the ribosome-mRNA complex to the surface of the rough ER where the protein's biogenesis will be completed. Ribosome-mRNA complexes that lack a signal sequence complete the translation of the mRNA—which encodes neither secretory nor membrane proteins—without attaching to the rough ER. For his work on signal sequences, Günter Blobel received the 1999 Nobel Prize for Physiology or Medicine. image N2-7


FIGURE 2-15 Synthesis and translocation of a secretory protein.


Günter Blobel

For more information about Günter Blobel and the work that led to his Nobel Prize, visit (accessed October 2014).

Why does the cell bother to segregate the synthesis of different protein populations to different cellular locales? Proteins that are destined either to reside in a membrane or to undergo secretion are inserted into or across the membrane of the rough ER at the same time that they are translated; this is called cotranslational translocation. As the nascent polypeptide chain emerges from the ribosome, it traverses the rough ER membrane and ultimately appears at the ER's luminal face. There, an enzyme cleaves the amino-terminal signal sequence while the protein is still being translocated. This is why proteins that are synthesized in vitro in the absence of membranes are longer than the same proteins that are produced by intact cells.

Simultaneous protein synthesis and translocation through the rough ER membrane requires machinery for signal recognition and protein translocation

The information embodied within a signal sequence explains how a nascent protein can direct a cell to complete that protein's translation at the time of translocation in the rough ER. However, the signal sequence by itself is not sufficient. Two critical pieces of targeting machinery are also necessary to direct the ribosome and its attached nascent peptide to the ER. The first is a ribonucleoprotein complex called the signal recognition particle (SRP), which binds to the signal sequence on the nascent peptide (see Fig. 2-15, stage 2). The SRP is composed of seven distinct polypeptides and a short strand of RNA. When the SRP binds to a nascent chain, it also binds a GTP molecule. The second vital piece of targeting machinery is a transmembrane component of the rough ER, the SRP receptor, also called the docking protein. Interaction between a signal sequence and the SRP, and subsequently between the SRP–nascent peptide–ribosome complex and the docking protein, directs the nascent chain to the rough ER's translocation apparatus.

Because the membrane of the rough ER has a finite number of docking sites, the cell must coordinate the synthesis of secretory and membrane proteins with the availability of docking sites. If all docking sites were occupied, and if the synthesis of nascent secretory and membrane proteins were allowed to continue unabated, these nascent peptides would be synthesized entirely in the cytoplasm on free ribosomes. As a consequence, these newly synthesized proteins would never arrive at their proper destination. The SRP serves as a regulatory system that matches the rate of synthesis of secretory and membrane proteins to the number of unoccupied translocation sites. By associating with a nascent signal sequence, the SRP causes the ribosome to halt further protein synthesis (see Fig. 2-15, stage 2). This state of translation arrest persists until the SRP–nascent peptide–ribosome complex finds an unoccupied docking protein with which to interact. Thus, SRP prevents secretory and membrane proteins from being translated until their cotranslational translocation can be ensured. Because SRP interacts only with nascent chains that bear signal sequences, ribosomes that synthesize proteins destined for release into the cytosol never associate with SRP, and their translation is never arrested. Thus, SRP serves as a highly specific spatial and temporal sorting machine, guaranteeing the accurate and efficient targeting of secretory and membrane proteins.

How does the cell terminate the translation arrest of the SRP–nascent peptide–ribosome complex? When this complex interacts with a docking protein (see Fig. 2-15, stage 3), one of the SRP's subunits hydrolyzes the previously bound GTP, thereby releasing the SRP from a successfully targeted nascent peptide–ribosome complex. In this way, the docking protein informs the SRP that its mission has been accomplished and it can return to the cytosol to find another ribosome with a signal peptide. A second GTP hydrolysis step transfers the nascent peptide from the docking protein to the actual translocation tunnel complex. GTP hydrolysis is a common event and is involved in the transmission of numerous cellular messages (see pp. 53 and 56). In this case, the two separate instances of GTP hydrolysis serve a quality-control function, because the activation of the GTPase activity depends on the delivery of the nascent peptide to the appropriate component in the translocation apparatus.

Adjacent to the docking protein in the membrane of the rough ER is a protein translocator termed a translocon (see Fig. 2-15, stage 3), which contains a tunnel through which the nascent protein will pass across the rough ER membrane. It appears that delivery of a nascent chain to the translocon causes the entrance of the translocator's tunnel, which is normally closed, to open. This opening of the translocon also allows the flow of small ions. The electrical current carried by these ions can be measured by the patch-clamp technique (see p. 154). By “gating” the translocon so that it opens only when it is occupied by a nascent protein, the cell keeps the tunnel's entrance closed when it is not in use. This gating prevents the Ca2+ stored in the ER from leaking into the cytoplasm.

Because the tunnel of the translocon is an aqueous pore, the nascent secretory or membrane protein does not come into contact with the hydrophobic core of the ER membrane's lipid bilayer during cotranslational translocation. Thus, this tunnel allows hydrophilic proteins to cross the membrane. As translation and translocation continue and the nascent protein enters the lumen of the rough ER, an enzyme called signal peptidase cleaves the signal peptide, which remains in the membrane of the rough ER (see Fig. 2-15, stage 4). Meanwhile, translation and translocation of the protein continue (see Fig. 2-15, stage 5). In the case of secretory proteins (i.e., not membrane proteins), the peptide translocates completely through the membrane. The ribosome releases the complete protein into the lumen of the rough ER and then dissociates from the rough ER (see Fig. 2-15, stage 6).

Proper insertion of membrane proteins requires start- and stop-transfer sequences

Unlike soluble proteins, nascent membrane proteins do not translocate completely through the membrane of the rough ER (Fig. 2-16A, stage 1). The hydrophobic amino-acid residues that will ultimately become the transmembrane segment of a membrane protein also function as a stop-transfer sequence (see Fig. 2-16A, stage 2). When a stop-transfer sequence emerges from a ribosome, it causes the translocon to open laterally, releasing the hydrophobic membrane-spanning segment into the hospitable environment of the rough ER membrane's hydrophobic core (see Fig. 2-16A, stage 3). In the meantime, the ribosomal machinery continues to translate the rest of the nascent protein. If the signal peptidase cleaves the amino terminus at this time, the end result is a protein with a single transmembrane segment, with the amino terminus in the lumen of the rough ER and the carboxyl terminus in the cytoplasm (see Fig. 2-16A, stage 4).


FIGURE 2-16 Synthesis of integral membrane proteins. A, Like a secreted protein, the membrane protein can have a cleavable signal sequence. In addition, it has a stop-transfer sequence that remains in the membrane as a membrane-spanning segment. B, The emerging protein lacks a signal sequence but instead has an internal start-transfer sequence, which is a bifunctional sequence that serves both as a signal sequence that binds SRPs and as a hydrophobic membrane-spanning segment. In this example, the positively charged region flanking the internal start-transfer sequence is on the carboxyl (C)–terminal end of the internal start-transfer sequence. Therefore, the C-terminal end is in the cytoplasm. C, The example is similar to that in B except that the positively charged region flanking the internal start-transfer sequence is on the amino- (N)–terminal end of the internal start-transfer sequence. D, The emerging peptide has alternating internal start-transfer and stop-transfer sequences.

There is another way of generating a protein with a single transmembrane segment. In this case, the protein lacks a signal sequence at the N terminus but instead has—somewhere in the middle of the nascent peptide—a bifunctional sequence that serves both as a signal sequence that binds SRP and as a hydrophobic membrane-spanning segment. This special sequence is called an internal start-transfer sequence. The SRP binds to the internal start-transfer sequence and brings the nascent protein to the rough ER, where the internal start-transfer sequence binds to the translocon in such a way that the more positively charged residues that flank the start-transfer sequence face the cytosol. Because these positively charged flanking residues can either precede or follow the hydrophobic residues of the internal start-transfer sequence, either the carboxyl (C) terminus or the N terminus can end up in the cytosol. If the more positively charged flanking residues are at the carboxyl-terminal end of the internal start-transfer sequence (see Fig. 2-16B), the protein will be oriented with its carboxyl terminus in the cytosol. If the more positively charged flanking residues are at the amino-terminal end of the internal start-transfer sequence (see Fig. 2-16C), the protein will be oriented with its amino terminus in the cytosol.

By alternating both stop-transfer sequences (see Fig. 2-16A) and internal start-transfer sequences (see Fig. 2-16B, C), the cell can fabricate membrane proteins that span the membrane more than once. Figure 2-16D shows how the cell could synthesize a multispanning protein with its N terminus in the cytosol. The process starts just as in Figure 2-16C, as the translation machinery binds to the rough ER (see Fig. 2-16D, stage 1) and the protein's first internal start-transfer sequence inserts into the translocon (see Fig. 2-16D, stage 2). However, when the first stop-transfer sequence reaches the translocon (see Fig. 2-16D, stage 3), the translocon disassembles, releasing the protein's first two membrane-spanning segments into the membrane of the rough ER. Note that the first membrane-spanning segment is the internal start-transfer sequence and the second is the stop-transfer sequence. In the meantime, an SRP binds to the second internal start-transfer sequence (see Fig. 2-16D, stage 4) and directs it to the rough ER (see Fig. 2-16D, stage 5) so that cotranslational translocation can once again continue (see Fig. 2-16D, stage 6). If there are no further stop-transfer sequences, the result will be a protein with three membrane-spanning segments.

Several points from the preceding discussion deserve special emphasis. First, translocation through the ER membrane can occur only cotranslationally. If a secretory or membrane protein were synthesized completely on a cytoplasmic ribosome, it would be unable to interact with the translocation machinery and consequently would not be inserted across or into the bilayer. As discussed below, this is not true for the insertion of either peroxisomal or mitochondrial proteins. Second, once a signal sequence emerges from a ribosome, there is only a brief period during which it is competent to mediate the ribosome's association with the ER and to initiate translocation. This time constraint is presumably due to the tendency of nascent polypeptide chains to begin to fold and acquire tertiary structure very soon after exiting the ribosome. This folding quickly buries hydrophobic residues of a signal sequence so that they cannot be recognized by the translocation machinery. Third, because the translocation channel appears to be fairly narrow, the nascent protein cannot begin to acquire tertiary structure until after it has exited at the ER's luminal face. Thus, the peptide must enter the translocation tunnel as a thin thread immediately after emerging from the ribosome. These facts explain why translocation is cotranslational. In systems in which post-translational translocation occurs (e.g., peroxisomes and mitochondria), special adaptations keep the newly synthesized protein in an unfolded state until its translocation can be consummated.

Finally, because the protein cannot flip once it is in the membrane, the scheme just outlined results in proteins that are inserted into the rough ER membrane in their final or “mature” topology. The number and location of a membrane protein's transmembrane segments, as well as its cytoplasmic and extracytoplasmic loops, are entirely determined during the course of its cotranslational insertion into the ER membrane. The order in which signal, internal start-transfer, and stop-transfer sequences appear in a membrane protein's primary structure completely determines how that protein will be arrayed across whatever membrane it ultimately comes to occupy.

Newly synthesized secretory and membrane proteins undergo post-translational modification and folding in the lumen of the rough ER

As a newly synthesized secretory or membrane protein exits the tunnel of the translocon and enters the lumen of the rough ER, it may undergo a series of post-translational modifications that will help it to acquire its mature conformation. The first alteration, as discussed above, is cleavage of the signal sequence (if present) and is accomplished very soon after the signal sequence has completed its translocation. Other covalent modifications that occur as translocation continues include glycosylation and formation of intramolecular disulfide bonds. Glycosylation refers here to the enzymatic, en-bloc coupling of preassembled, branched oligosaccharide chains that contain 14 sugar molecules (Fig. 2-17A) to asparagine residues that appear in the sequence Asn-X-Ser or Asn-X-Thr (X can be any amino acid except proline). These N-linked sugars (N is the single-letter amino-acid code for asparagine) will go on to be extensively modified as the protein passes through other organellar compartments. The addition of sugar groups to proteins can serve numerous functions, which include increasing the protein's stability and endowing it with specific antigenic, adhesive, or receptor properties.


FIGURE 2-17 Post-translational modifications of integral membrane proteins. A, An enzyme in the ER lumen attaches a preassembled, branched, oligosaccharide chain to an asparagine (Asn or N) residue on the nascent protein. B, An enzyme in the ER lumen cleaves the protein and couples the protein's new terminal carboxyl group to the terminal amino group on the GPI molecule.

Disulfide bond formation is catalyzed by protein disulfide isomerase, an enzyme that is retained in the ER lumen through noncovalent interactions with ER membrane proteins. Because the cytoplasm is a reducing environment, disulfide bonds can form only between proteins or protein domains that have been removed from the cytosolic compartment through translocation to the ER's interior. Other, more specialized modifications also take place in the lumen of the rough ER. For example, the ER contains the enzymes responsible for the hydroxylation of the proline residues that are present in newly synthesized collagen chains.

The ER also catalyzes the formation of GPI linkages to membrane proteins (see Fig. 2-17B). GPI-linked proteins are synthesized as transmembrane polypeptides, with a typical membrane-spanning region. Shortly after their translation, however, their lumen-facing domains are cleaved from the membrane-spanning segments and covalently transferred to the GPI phospholipid. They retain this structure and orientation throughout the remainder of their journey to the cell surface. A defect in the synthesis of GPI-linked proteins underlies the human disease paroxysmal nocturnal hematuria (Box 2-1).

Box 2-1

Paroxysmal Nocturnal Hematuria

The list of proteins embedded in the plasma membrane via a GPI linkage is remarkably long and ever growing. In red blood cells, the inventory of GPI-linked proteins includes a pair of polypeptides, decay-accelerating factor (DAF) and CD59, which help protect the erythrocytes from being accidentally injured by constituents of the immune system. One of the mechanisms that the immune system uses to rid the body of invading bacteria involves the activation of the complement cascade. Complement is a complex collection of proteins that circulate in the blood plasma. The complement system recognizes antibodies that are bound to the surface of a bacterium or polysaccharides in the bacterial membrane. This recognition initiates a cascade of enzymatic cleavages that results in the assembly of a subset of complement proteins to form the membrane attack complex, which inserts itself into the membrane of the target organism and forms a large pore that allows water to rush in (see pp. 109–110). The target bacterium swells and undergoes osmotic lysis. Unfortunately, the complement system's lethal efficiency is not matched by its capacity to discriminate between genuine targets and normal host cells. Consequently, almost every cell type in the body is equipped with surface proteins that guard against a misdirected complement attack.

DAF and CD59 are two such proteins that interfere with distinct steps in the complement activation pathway. Because GPI linkages couple both proteins to the membrane, any dysfunction of the enzymes that participate in the transfer of GPI-linked proteins from their transmembrane precursors to their GPI tails in the ER would interfere with the delivery of DAF and CD59 to their sites of functional residence at the cell surface. One of the proteins that participates in the synthesis of the GPI anchor is a sugar transferase encoded by the phosphatidylinositol glycan class A (PIG-A) gene. This gene is located on the X chromosome. Because every cell has only one working copy of the X chromosome (although female cells are genetically XX, one of the two X chromosomes is inactivated in every cell), if a spontaneous mutation occurs in the PIG-A gene in a particular cell, that cell and all of its progeny will lose the ability to synthesize GPI-linked proteins.

In paroxysmal nocturnal hemoglobinuria (i.e., the appearance of hemoglobin in the urine at night, with a sharp onset), a spontaneous mutation occurs in the PIG-A gene in just one of the many precursor cells that give rise to erythrocytes. All of the erythrocytes that arise from this particular precursor, therefore, are deficient in GPI-linked protein synthesis. Consequently, these cells lack DAF and CD59 expression and are susceptible to complement attack and lysis. For reasons that are largely unknown, the complement system is somewhat more active during sleep, so the hemolysis (lysis of erythrocytes) occurs more frequently at night in these patients. Some of the hemoglobin released by this lysis is excreted in the urine.

Because the PIG-A gene product is required for the synthesis of all GPI-linked proteins, the plasma membranes of affected red blood cells in patients with paroxysmal nocturnal hemoglobinuria are missing a number of different proteins that are found in the surface membranes of their normal counterparts. It is the lack of DAF and CD59, however, that renders the cells vulnerable to complement-mediated killing and that creates the symptoms of the disease. Paroxysmal nocturnal hemoglobinuria is an uncommon disease. Because it is the result of an acquired mutation, it is much more likely to occur in people of middle age than in children. Patients with paroxysmal nocturnal hemoglobinuria are likely to become anemic and can suffer life-threatening disorders of clotting and bone marrow function. It is a chronic condition, however, and more than half of patients survive at least 15 years after diagnosis.

Perhaps the most important maturational process for a nascent chain emerging into the ER lumen is the acquisition of tertiary structure. The folding of a secretory or membrane protein is determined during and immediately after its cotranslational translocation. The progress of protein folding influences—and is influenced by—the addition of sugar residues and the formation of disulfide bridges. Proteins fold into conformations that minimize their overall free energies. Their extramembranous surfaces are composed of hydrophilic residues that interact easily with the aqueous solvent. Hydrophobic residues are hidden in internal globular domains where they can be effectively isolated from contact with water or charged molecules. Left to its own devices, a linear strand of denatured protein will spontaneously fold to form a structure that reflects these thermodynamic considerations. Thus, protein folding requires no catalysis and can occur without help from any cellular machinery. However, the cell is not content to allow protein folding to follow a random course and instead orchestrates the process through the actions of molecular chaperones.

The chaperones constitute a large class of ATP-hydrolyzing proteins that appear to participate in a wide variety of polypeptide-folding phenomena, including the initial folding of newly synthesized proteins as well as the refolding of proteins whose tertiary structures have been damaged by exposure to high temperature (i.e., heat shock) or other denaturing conditions. Chaperones bind to unfolded protein chains and stabilize them in an unfolded conformation, thus preventing them from spontaneously folding into what might be an energetically favorable but biologically useless arrangement. Using energy that is provided through ATP hydrolysis, the chaperones sequentially release domains of unfolded proteins and thus allow them to fold in an ordered fashion. An excess of unfolded or misfolded proteins causes ER stress, triggering the unfolded protein response. image N2-8 Distinct subclasses of chaperones are present in several cell compartments, including the cytoplasm, the mitochondrion, and the lumen of the rough ER. Newly synthesized secretory and membrane proteins interact with ER chaperones as they exit from the tunnel of the translocon and subsequently disengage from the chaperones to assume their mature tertiary structure.


Unfolded Protein Response

Contributed by D. Narayan Rao

In response to ER stress due to the accumulation of unfolded proteins in the lumen, the unfolded protein response (URP) acts to restore ER homeostasis. URP activation has three unique mechanisms that operate in parallel

1. Feedback control: regulating the rate of protein synthesis by temporarily halting protein translation

2. Cell fate regulation: recognizing and eliminating misfolded proteins

3. Adaptive response: ramping up production of molecular chaperones involved in protein folding


Oslowski CM, Urano F. Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Conn PM. Methods in Enzymology. Academic Press: San Diego; 2011:71–92 [Accessed July 24, 2014].

The acquisition of tertiary structure is followed quickly by the acquisition of quaternary structure. As noted above in this chapter, many membrane proteins assemble into oligomeric complexes in which several identical or distinct polypeptides interact with one another to form a macromolecular structure. Assembly of these multimers generally occurs in the ER. It is unknown whether the oligomeric assembly process occurs entirely spontaneously or if, like folding, it is orchestrated by specialized cellular mechanisms. Cells clearly go to great trouble to ensure that proteins inserted into or across their ER membranes are appropriately folded and oligomerized before allowing them to continue with their postsynthetic processing. As discussed below, proteins destined for secretion from the cell or for residence in plasma or organellar membranes depart the ER for further processing in the membranous stacks of the Golgi complex. This departure is entirely contingent on successful completion of the protein folding and assembly operations.

Misfolded or unassembled proteins are retained in the ER and ultimately degraded. The ER chaperone proteins play a critical role both in identifying proteins with incorrect tertiary or quaternary structures and in actively preventing their egress to the Golgi complex. Proteins that have not folded or assembled correctly are destroyed through a process known as ERAD (endoplasmic reticulum–associated degradation). The sequential covalent addition of ubiquitin monomers results in the formation of a branched-chain ubiquitin polymer that marks these proteins for destruction. Ubiquitin is a small protein of 76 amino-acid residues. The process known as retrotranslocation removes ubiquitin-tagged proteins from the ER membrane, and a large cytoplasmic complex of proteolytic enzymes—the proteasome—degrades the ubiquitinated proteins.

Secretory and membrane proteins follow the secretory pathway through the cell

The rough ER is the common point of origin for the cell's secretory and membrane proteins. Most of these proteins are not retained in the rough ER but depart for distribution to their sites of ultimate functional residence throughout the cell. As is true for their arrival in the rough ER, the departure of these proteins is a highly organized and regimented affair. In fact, the rough ER is the first station along the secretory pathway, which is the route followed (at least in part) by all secretory and membrane proteins as they undergo their post-translational modifications (Fig. 2-18).


FIGURE 2-18 The secretory pathway. After their synthesis in the rough ER, secretory and membrane proteins destined for the plasma membrane move through the Golgi stacks and secretory vesicles. In the constitutive pathway, vesicles fuse spontaneously with the plasma membrane. In the regulated pathway, the vesicles fuse only when triggered by a signal such as a hormone.

The elucidation of the secretory pathway occurred in the 1960s, mainly in the laboratory of George Palade. For his contribution, Palade was awarded the 1975 Nobel Prize in Physiology or Medicine. image N2-9 This work also exploited the unique properties of pancreatic acinar cells to illuminate the central features of the biogenesis of secretory proteins. Because ~95% of the protein that is synthesized by pancreatic acinar cells is digestive enzymes destined for secretion (see p. 882), when these cells are fed radioactively labeled amino acids, the majority of these tracer molecules are incorporated into secretory polypeptides. Within a few minutes after the tracer is added, most of the label is associated with a specialized subregion of the rough ER. Known as transitional zones, these membranous saccules are ribosome studded on one surface and smooth on the opposite face (see Fig. 2-18). The smooth side is directly apposed to one pole of the pancake-like membrane stacks (or cisternae) of the Golgi complex. Smooth-surfaced carrier vesicles crowd the narrow moat of cytoplasm that separates the transitional zone from the Golgi. These vesicles “pinch off” from the transitional zone and fuse with a Golgi stack. From this first or cis-Golgi stack, carrier vesicles ferry the newly synthesized proteins sequentially and vectorially through each Golgi stack, ultimately delivering them to the trans-most saccule of the Golgi. Finally, the newly synthesized secretory proteins appear in secretory vesicles (also called secretory granules in many tissues).


George Palade

For more information about George Palade and the work that led to his Nobel Prize, visit (accessed October 2014).

The journey from the rough ER to the secretory vesicle takes ~45 minutes in pancreatic acinar cells and requires the expenditure of metabolic energy. Each nucleated eukaryotic cell possesses a secretory pathway that shares this same general outline, although the specific features reflect the cell's particular function. The secretory pathway of the pancreatic acinar cell, for example, is specially adapted to accommodate the controlled secretion of protein via the so-called regulated pathway. Instead of being released from the cell continuously as they are produced, newly synthesized secretory proteins are held in specialized secretory vesicles that serve as an intracellular storage depot. This type of storage occurs in several cells, including those of endocrine and exocrine secretory tissues, and neurons. When the cells receive the requisite message, the storage vesicles fuse with the plasma membrane, sometime at a specialized structure called a porosome, in a process known as exocytosis. image N2-10 The vesicles then dump their contents into the extracellular space. In the case of the pancreatic acinar cells, the enzymes are secreted into the pancreatic ductules and then make their way to the site of digestion in the duodenum (see p. 881).


The Porosome

Contributed by Bhanu P. Jena

Porosomes are the universal secretory machinery in cells. In the past decade, this newly discovered cellular structure at the cell plasma membrane, measuring only a few nanometers, has provided a molecular understanding of the secretory process in cells. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicular contents to the outside during cell secretion. The mouth of the porosome opening to the outside ranges in size from 150 nm in diameter in acinar cells of the exocrine pancreas to 12 nm in neurons. The mouth dilates during cell secretion, returning to its resting size following completion of the process (eFig. 2-2).


EFIGURE 2-2 Porosome: the secretory portal in mammalian cells. A, High-resolution atomic force micrograph showing a single pit with four 100- to 180-nm porosomes within (blue arrowhead) at the apical plasma membrane in a live pancreatic acinar cell. B, Electron micrograph depicting a porosome (blue arrowhead) close to a microvillus (MV) at the apical plasma membrane (PM) of a pancreatic acinar cell. Note the association of the porosome membrane (yellow arrowhead) and the zymogen granule membrane (ZGM; red arrowhead) of a docked zymogen granule (ZG; inset). Cross section of a circular complex at the mouth of the porosome is seen (blue arrowhead). POM, Porosome membrane. C, Schematic diagram of pits (yellow arrow) and porosomes (blue arrow) at the cell plasma membrane (PM). ZGs are the secretory vesicles in acinar cells of the exocrine pancreas that transiently dock and fuse at the porosome base to expel intravesicular contents during secretion. D, Several porosomes within a pit shown at time 0, 5 minutes, and 30 minutes (from left to right) following stimulation of secretion. Section analysis across three porosomes is shown, with the blue arrowhead pointing at a porosome. Note the dilation of the porosome at the 5-minute time point and its return to near resting size after 30 minutes following stimulation of secretion. E, Percentage of total cellular amylase release in the presence (yellow bars) and absence (blue bars) of the secretagogue Mas7. Note the increase in porosome diameter in D, correlating with an increase in total cellular amylase release at 5 minute following stimulation of secretion. At 30 minutes following a secretory stimulus, there is a decrease in porosome diameter (D) and no further increase in amylase secretion beyond that at the 5-minute time point. No significant changes in amylase secretion or porosome diameter were observed in control cells in either the presence or absence of the nonstimulatory mastoparan analog (Mas17). F, Electron micrograph of a porosome (blue arrowheads) at the nerve terminal in association with a synaptic vesicle (SV) at the presynaptic membrane (Pre-SM). Notice the central plug-like structure at the neuronal porosome opening. post-SM, postsynaptic membrane. G, Atomic force micrograph of a neuronal porosome in physiological buffer, also showing the central plug (blue arrowhead) at its opening. The central plug in the neuronal porosome complex may regulate its rapid close-open conformation during neurotransmitter release. The neuronal porosome is an order of magnitude smaller (10 to 15 nm) than the porosome in the exocrine pancreas (100 to 180 nm). Note the central plug and eight interconnected ridges within the porosome complex. H, Electron-density maps of negatively stained electron micrographs of isolated neuronal porosome protein complex. Note the ~12-nm complex exhibiting a circular profile and having a central plug, with eight interconnected protein densities at the rim of the complex. Bar = 5 nm. I, Atomic force micrograph of a pit and three porosomes within (one indicated by the blue arrowhead) in a pancreatic acinar cell and the specific immunolocalization of amylase-specific immunogold (yellow spots) demonstrating amylase secretion through the structure. J, Electron micrograph of a liposome-reconstituted porosome complex isolated from a pancreatic acinar cell. Note the cup-shaped, basket-like morphology of the porosome complex reconstituted in a 500-nm lipid vesicle. Bar = 100 nm. K, The lipid bilayer–reconstituted porosome complex is functional. Top panel shows a schematic drawing of the EPC 9 setup for making electrophysiological measurements. Isolated zymogen granules (ZG) added to the cis compartment of the bilayer chamber dock and fuse with the reconstituted porosomes at the bilayer and are detected as an increase in capacitance and current activity and as a concomitant time-dependent release of amylase to the trans compartment of the bilayer chamber as determined using immunoblot assay. (From Jena BP: Functional organization of the porosome complex and associated structures facilitating cellular secretion. Physiology 24:367–376, 2009.)

The past decade has witnessed the elucidation of the composition of the porosome, as well as its structure, its dynamics at nanometer resolution and in real time, and its functional reconstitution into artificial lipid membranes. Experiments have also demonstrated the molecular mechanism of secretory vesicle fusion at the porosome base as well as secretory vesicle swelling enabling expulsion of intravesicular contents. It has become clear that secretory vesicles transiently dock, fuse, partially expel their contents, and dissociate—a process that allows multiple rounds of docking-fusion-expulsion-dissociation to occur. It has been further determined that swelling of secretory vesicles is required for the expulsion of intravesicular contents during cell secretion, and the extent of swelling is directly proportional to the amount of vesicular contents expelled. These findings have led to a paradigm shift in our understanding of cell secretion, resolving the longstanding conundrum regarding the generation of partially empty vesicles as seen in electron micrographs following cell secretion.


Cho SJ, Jeftinija K, Glavaski A, et al. Structure and dynamics of the fusion pores in live GH-secreting cells revealed using atomic force microscopy. Endocrinology. 2002;143:1144–1148.

Jena BP. Functional organization of the porosome complex and associated structures facilitating cellular secretion. Physiology. 2009;24:367–376.

Jena BP, Schneider SW, Geibel JP, et al. Gi regulation of secretory vesicle swelling examined by atomic force microscopy. Proc Natl Acad Sci U S A. 1997;94:13317–13322.

Jeremic A, Kelly M, Cho SJ, et al. Reconstituted fusion pore. Biophys J. 2003;85:2035–2043.

Most cell types, however, deliver newly synthesized secretory and membrane proteins to the cell surface in a continuous and unregulated fashion, which is referred to as the constitutive pathway. Specialized cells that have the capacity for regulated delivery also send an important subset of their secretory and membrane protein synthetic products to the cell surface constitutively. The regulated and constitutive secretory pathways are identical except for the final station of the Golgi complex. At this point, the “regulated” proteins divert to the specialized secretory vesicles described in the previous paragraph. The “constitutive” proteins, at the trans-most cisterna of the Golgi complex, sort into other secretory vesicles, which move directly to the cell surface. There, the constitutive membrane proteins are delivered to the plasma membrane, and the constitutive secretory proteins are immediately exocytosed.

This section has provided a broad overview of the secretory pathway. In the following sections, we examine the details of how newly synthesized proteins move between organellar compartments of the secretory pathway, how the proteins are processed during this transit, and how they are sorted to their final destination.

Carrier vesicles control the traffic between the organelles of the secretory pathway

As the preceding discussion suggests, the secretory pathway is not a single, smooth, continuous highway but rather a series of saltatory translocations from one discrete organellar compartment to the next. Each of these steps requires some orchestration to ensure that the newly synthesized proteins arrive at their next terminus.

The cell solves the problem of moving newly synthesized proteins between membranous organelles by using membrane-enclosed carrier vesicles (or vesicular carriers). Each time proteins are to be moved from one compartment to the next, they are gathered together within or beneath specialized regions of membrane that subsequently evaginate or pinch off to produce a carrier vesicle (see Fig. 2-18). Secretory proteins reside within the lumen of the carrier vesicle, whereas membrane proteins span the vesicle's own encapsulating bilayer. On arrival at the appropriate destination, the carrier vesicle fuses with the membrane of the acceptor organelle, thus delivering its contents of soluble proteins to the organelle's lumen and its cargo of membrane proteins to the organelle's own membrane. Carrier vesicles mediate the transport of secretory and membrane proteins across the space between the ER's transition zone and the cis-Golgi stack and also between the rims of the Golgi stacks themselves. Often assisting the movement between one vesicular compartment and the next are the cytoskeleton and molecular motors. image N2-4

A few critical facts deserve emphasis. First, throughout the formation, transit, and fusion of a carrier vesicle, no mixing occurs between the vesicle lumen and cytosol. The same principle applies to the carrier vesicle's membrane-protein passengers, which were inserted into the membrane of the rough ER with a particular topology. Those domains of a membrane protein that are exposed to the cytosol in the rough ER remain exposed to the cytosol as the protein completes its journey through the secretory pathway.

Second, the flow of vesicular membranes is not unidirectional. The rate of synthesis of new membrane lipid and protein in the ER is less than the rate at which carrier vesicles bud off of the ER that is bound for the Golgi. Because the sizes of the ER and Golgi are relatively constant, the membrane that moves to the Golgi via carrier vesicles must return to the ER. This return is again accomplished by vesicular carriers. Each discrete step of the secretory pathway must maintain vesicle-mediated backflow of membrane from the acceptor to the donor compartment so that each compartment can retain a constant size.

Finally, we have already noted that each organelle along the secretory pathway is endowed with a specific set of “resident” membrane proteins that determines the properties of the organelle. Despite the rapid forward and backward flow of carrier vesicles between successive stations of the secretory pathway, the resident membrane proteins do not get swept along in the flow. They are either actively retained in their home organelles' membranes or actively retrieved by the returning “retrograde” carrier vesicles. Thus, not only the size but also the composition of each organelle of the secretory pathway remains essentially constant despite the rapid flux of newly synthesized proteins that it constantly handles.

Specialized protein complexes, such as clathrin and coatamers, mediate the formation and fusion of vesicles in the secretory pathway

The formation of a vesicle through evagination appears to be geometrically indistinguishable from its fusion with a target membrane. In both cases, a cross-sectional view in the electron microscope reveals an “omega” profile, which is so named because the vesicle maintains a narrow opening to the organellar lumen that resembles the shape of the Greek letter omega (Ω). However, different problems are confronted during the formation and fusion of membrane vesicles.

Vesicle Formation in the Secretory Pathway

To form a spherical vesicle from a planar membrane, the mechanism that pulls the vesicle off from the larger membrane must grab onto the membrane over the entire surface of the nascent vesicle. The mechanism that achieves this makes use of a scaffold that is composed of coat proteins. The cell has at least two and probably more varieties of coat proteins. The best characterized of these is clathrin, which mediates the formation of secretory vesicles from the trans Golgi. Clathrin also mediates the internalization of membrane from the cell surface during the process of endocytosis, which is the reverse of exocytosis. Another major protein coat that is involved in trafficking of vesicles between the ER and Golgi, and between the stacks of the Golgi, is a protein complex known as coatamer. Both clathrin and coatamer proteins form the borders of a cage-like lattice.

In the case of clathrin, the coat proteins preassemble in the cytoplasm to form three-armed “triskelions” (Fig. 2-19A). A triskelion is not planar but resembles the three adjoining edges of a tetrahedron. As triskelions attach to one another, they produce a three-dimensional structure resembling a geodesic dome with a roughly spherical shape. A triskelion constitutes each vertex in the lattice of hexagons and pentagons that form the cage.


FIGURE 2-19 Vesicle formation and fusion. A, Clathrin mediates the formation of secretory vesicles that bud off from the trans Golgi as well as the internalization of membrane from the cell surface during the process of endocytosis. B, A complex of proteins forms a bridge between the vesicle and the target membranes. ATP provides the fuel for fusion. The Rab appears to be a molecular switch.

The triskelions of clathrin attach indirectly to the surface of the membrane that is to be excised by binding to the cytosolic tails of membrane proteins. Mediating this binding are adapter proteins, called adaptins, that link the membrane protein tails to the triskelion scaffold. The specificity for particular membrane proteins is apparently conferred by specialized adaptins. Triskelions assemble spontaneously to form a complete cage that attaches to the underlying membrane and pulls it up into a spherical configuration. Completion of the cage occurs simultaneously with the pinching off of the evaginated membrane from the planar surface, forming a closed sphere.

The pinching off, or fission, process involves the action of a GTP-binding protein called dynamin, which forms a collar around the neck of the forming vesicle and plays a role in severing the neck. The fission process must include an intermediate that resembles the structure depicted in Figure 2-19A. According to the prevalent view, each of the lumen-facing leaflets of membrane lipids fuse, leaving only the cytoplasmic leaflets to form a continuous bridge from the vesicle to the donor membrane. This bridge then breaks, and fission is complete.

Once formed, the clathrin-coated vesicle cannot fuse with its target membrane until it loses its cage, which prevents the two membranes from achieving the close contact required to permit fusion. Because formation of the clathrin cage is spontaneous and energetically favorable, dissolution of the cage requires energy. Uncoating is accomplished by a special class of cytoplasmic enzymes that hydrolyze ATP and use the energy thus liberated to disassemble the scaffold (see Fig. 2-19A).

The function of coatamers is similar to that of clathrin in that coatamers form a cage around a budding membrane. However, coatamer coats differ from clathrin cages in several respects. First, coatamer coats are composed of several coatamer proteins (COPs), one of which is related to the adaptins. Second, unlike the spontaneous assembly of the clathrin triskelions, assembly of the coatamer coat around the budding vesicle requires ATP. Third, a coatamer-coated vesicle retains its coat until it docks with its target membrane.

Vesicle Fusion in the Secretory Pathway

Membrane fusion occurs when the hydrophobic cores of two bilayers come into contact with one another, a process that requires the two membranes to be closely apposed. Because the cytoplasmic leaflets of most cellular membranes are predominantly composed of negatively charged phospholipids, electrostatic repulsion prevents this close apposition from occurring spontaneously. To overcome this charge barrier, and to assist in targeting as well, a multicomponent protein complex forms and acts as a bridge, linking vesicular membrane proteins to membrane proteins in the target bilayer (see Fig. 2-19B). Investigators have established the components of this complex by use of three approaches: studies of the membrane-fusion steps involved in vesicular transport between successive Golgi stacks, genetic analysis of protein secretion in yeast, and molecular dissection of the protein constituents of the synaptic vesicles of nerve terminals. In each case, the same proteins are instrumental in attaching the donor and target membranes to one another.

The central components of the bridge are proteins known as SNAREs (so named because they act as receptors for the SNAPs discussed in the next paragraph). There are SNAREs in both the vesicular membrane (v-SNAREs) and the membrane of the target organelle (t-SNAREs). Typical of the SNARE family members are those that participate in the fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane of the axons in neurons (see pp. 219–221). In that setting, the v-SNARE is known as synaptobrevin, and proteins known as syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) together act as t-SNAREs. The t-SNAREs and v-SNAREs bind to each other extremely tightly, pulling the vesicular and target membranes close together. This proximity alone may be sufficient to initiate fusion, although this point remains controversial. In cells that employ rapid and tightly regulated membrane fusion, such as neurons, a protein called synaptotagmin, which associates with the SNARE fusion complex, senses an increase in the cytoplasmic concentration of Ca2+ and triggers fusion (see pp. 219–221). Although the nature of the fusion event itself remains unclear, clues have emerged about its regulation. Fusion requires the participation of a class of small GTP-binding proteins called Rabs that are important for signaling. Rabs appear to act as molecular switches that assemble with the SNARE fusion complex when they are binding GTP but dissociate from the complex after they hydrolyze the GTP to GDP. Rab-GTP appears to play a critical role in regulating the activity of the fusion complex. Numerous Rab isoforms exist, each isoform associated with a different vesicular compartment and a distinct membrane-to-membrane translocation step.

Once fusion occurs, the former vesicle generally loses its spherical shape rapidly as it becomes incorporated into the target membrane. This “flattening out” is the result of surface tension, inasmuch as the narrow radius of curvature demanded by a small spherical vesicle is energetically unfavorable. After fusion, it is also necessary to disassemble the v-SNARE/t-SNARE complex so that its components can be reused in subsequent fusion events. The dissociation step involves the activity of two additional components that participate in the SNARE complex. The first is an ATP-hydrolyzing enzyme; because it is inhibited by the alkylating agent N-ethylmaleimide (NEM), it was named NEM-sensitive factor (NSF). Soluble NSF attachment proteins (the SNAPs mentioned above), which target NSF to the SNARE complex, are the second. Hydrolysis of ATP by NSF causes dissociation of the SNARE complex, thus regenerating the fusion machinery. Homologs of the neuronal t-SNARE and v-SNARE proteins are found in almost every cell type in the body and are thought to participate in most if not all membrane fusion events.

Newly synthesized secretory and membrane proteins are processed during their passage through the secretory pathway

While in the rough ER, newly synthesized secretory and membrane proteins undergo the first in a series of post-translational modifications (see pp. 32–34), including glycosylation, disulfide bond formation, and the acquisition of tertiary structure. On delivery to the cis stack of the Golgi complex, these proteins begin a new phase in their postsynthetic maturation. For many proteins, the most visible byproduct of this second phase is the complete remodeling of their N-linked sugar chains, originally attached in the rough ER.

Of the 14 sugar residues transferred en bloc to newly synthesized proteins during N-linked glycosylation (see p. 32), nine are mannose and three are glucose (Fig. 2-20A). Enzymes called glucosidases and one called a mannosidase associate with the luminal face of the ER and remove the three glucose residues and one mannose. This trimming process is a critical step in the quality-control process through which the cell determines whether a protein is properly folded and ready to proceed to subsequent stations of the secretory pathway. An enzyme called uridine diphosphate (UDP)–glucose glycoprotein glucosyltransferase (UGGT) inspects proteins in the lumen of the ER. If they are not appropriately folded, UGGT adds a single glucose residue to the trimmed sugar tree. Proteins bearing this single glucose residue bind calnexin or calreticulin, ER chaperone proteins that retain misfolded proteins until they fold correctly or become targeted for degradation.


FIGURE 2-20 Modification and assembly of the sugar chains on proteins in the Golgi. A, Remodeling of N-linked sugars. B, Proteoglycans. A trisaccharide links glycosaminoglycan chains to the protein via the –OH group of a serine residue. The glycosaminoglycan is made up of n repeating disaccharide units, one of which is always an amino sugar.

As proteins arrive from the ER, mannosidases in the cis Golgi attack the N-linked sugar trees, shearing off all except two N-acetylglucosamine and five mannose residues. As the proteins pass from the cis-Golgi cisterna to the medial cisterna and ultimately to the trans-Golgi cisterna, another mannosidase removes two additional mannose residues, and other enzymes add sugars to the stump of the original sugar tree in a process referred to as complex glycosylation.

The addition of new sugars occurs one residue at a time and is accomplished by enzymes called sugar transferases that face the lumens of the Golgi stacks. Each sugar is transported from the cytoplasm to the Golgi lumen by a carrier protein that spans the Golgi membrane. Throughout the maturation process, the N-linked sugar chains are always exposed only to the luminal face of the Golgi. image N2-11


Sugar Uptake into the Golgi

Contributed by Michael Caplan

The attachment of a sugar molecule to the growing N-linked sugar chain occurs in a series of four steps:

Step 1: In the cytosol, the sugar is covalently coupled to a nucleoside diphosphate (either UDP or GDP, depending on the sugar to be transported). The result of this reaction is a sugar nucleoside diphosphate (e.g., UDP-galactose). (An exception to this rule is sialic acid, in which the sugar is coupled to cytidine monophosphate [CMP], a nucleoside monophosphate, rather than a diphosphate.)

Step 2: A carrier protein in the membrane of the Golgi moves the sugar nucleoside diphosphate from the cytoplasm to the lumen of the Golgi.

Step 3: The sugar transferases use the sugar nucleoside diphosphate (e.g., UDP-galactose) as a substrate by catalyzing a reaction that couples the sugar residue (e.g., galactose) to the growing N-linked chain. As a byproduct, this reaction generates a nucleoside diphosphate (e.g., UDP), which is then converted to a nucleoside monophosphate (uridine monophosphate [UMP]) plus inorganic phosphate.

Step 4: The same carrier protein that imports the sugar nucleoside diphosphate (e.g., UDP-galactose) exports the nucleoside monophosphate (e.g., UMP) that is the byproduct of the above transferase reaction. Because the carrier protein simultaneously imports the sugar nucleoside diphosphate and exports the nucleoside monophosphate, this carrier protein is an example of an exchanger (see pp. 123–125).

Each cisterna of the Golgi is characterized by a different set of sugar transferases and sugar transporters. Thus, each Golgi compartment catalyzes a distinct step in the maturation of the N-linked chains. Complex glycosylation, therefore, proceeds like an assembly line from one modification station to the next. Because proteins have different shapes and sizes, however, the degree to which a sugar chain of any given polypeptide has access to each transferase can vary quite extensively. Thus, each protein emerges from the assembly line with its own particular pattern of complex glycosylation. The Golgi's trans-most cisterna houses the enzymes responsible for adding the terminal sugars, which cap the N-linked chain. The final residue of these terminal sugars is frequently N-acetylneuraminic acid, also known as sialic acid. At neutral pH, sialic acid is negatively charged. This acidic sugar residue therefore is responsible for the net negative electrostatic charge that is frequently carried by glycoproteins.

The Golgi's function is not limited to creating N-linked sugar tree topiaries. It oversees a number of other post-translational modifications, including the assembly of O-linked sugars. Many proteins possess O-linked sugar chains, which attach not to asparagine residues but to the hydroxyl groups (hence, O) of serine and threonine residues. The O-linked sugars are not preassembled for en-bloc transfer the way that the original 14-sugar tree is added in the rough ER in the case of their N-linked counterparts. Instead, the O-linked sugars are added one residue at a time by sugar transferases such as those that participate in the remodeling of complex N-linked glycosylation. O-linked chains frequently carry a great deal of negatively charged sialic acid.

Proteoglycans contain a very large number of a special class of O-linked sugar chains that are extremely long (see Fig. 2-20B). Unlike other O-linked sugars that attach to the protein core via an N-acetylglucosamine, the sugar chain in a proteoglycan attaches via a xylose-containing three-sugar “linker” to a serine residue on the protein backbone. One or more glycosaminoglycan side chains are added to this linker, one sugar at a time, to form the mature proteoglycan.

As the sugar chains grow, enzymes can add sulfate groups and greatly increase the quantity of negative charge that they carry. Sulfated proteoglycans that are secreted proteins become important components of the extracellular matrix and are also constituents of mucus. Proteoglycan chains can also be attached to membrane proteins that eventually reach the plasma membrane. The negatively charged sugars that are associated with the glycosaminoglycan groups, which are present both in mucus and on cell surface glycoproteins, can help form a barrier that protects cells from harsh environmental conditions such as those inside the stomach (see p. 874). In the upper portion of the respiratory tract, the mucus assists in the removal of foreign bodies (see p. 597).

Newly synthesized proteins are sorted in the trans-Golgi network

From their common point of origin at the rough ER, newly synthesized secretory and membrane proteins must be distributed to a wide variety of different subcellular destinations. How can a cell recognize an individual protein from among the multitudes that are inserted into or across the membranes of the rough ER and ensure its delivery to the site of its ultimate functional residence? Such a sorting operation has two prerequisites: (1) each protein to be sorted must carry some manner of address or “sorting signal” that communicates its destination, and (2) the cell must possess machinery capable of reading this sorting signal and acting on the information it embodies.

For many proteins, sorting occurs in the trans-Golgi network (TGN). The trans-most cisterna of the Golgi complex is morphologically and biochemically distinct from the other Golgi stacks. Viewed in cross section, it appears as a complex web of membranous tubules and vesicles (Fig. 2-21). This structure befits the TGN's apparent function as a staging area from which carrier vesicles depart to transport their specific protein cargoes to one of many distinct subcellular locales.


FIGURE 2-21 Sorting of lysosomal enzymes.

Sorting machinery within or at the TGN appears to segregate classes of proteins—each bound for a common destination—into small discrete clusters. Each cluster is subsequently incorporated into a separate carrier vesicle, which evaginates from the TGN membrane and mediates the final stage of delivery. In the case of secretory proteins, this clustering happens within the lumen of the TGN. In fact, such clusters of secretory proteins can be directly visualized in the electron microscope. Membrane proteins gather into two-dimensional clusters in the plane of the TGN membrane. Carrier vesicles incorporate these clusters into their own bilayers. Proteins bound for different destinations co-cluster in different subdomains of the TGN. Secretory and membrane proteins that are earmarked for the same destination can cluster in the same subdomain of the TGN and can be incorporated into the same carrier vesicle. Therefore, the TGN appears to function as a cellular transportation terminal that is able to direct groups of passengers who are carrying the same tickets to a common waiting area and ultimately to load them onto a common shuttle. Ticket agents herd passengers bearing different tickets into different waiting lounges.

A mannose-6-phosphate recognition marker is required to target newly synthesized hydrolytic enzymes to lysosomes

One of the most thoroughly established sorting paradigms is the pathway for newly synthesized lysosomal enzymes. Like secretory proteins, lysosomal enzymes carry amino-terminal signal sequences that direct their cotranslational translocation across the membrane of the rough ER. Their N-linked glycosylation and folding proceed in the usual fashion, after which they join all of the other simultaneously synthesized proteins in the Golgi complex (see Fig. 2-21, stage 1).

A special sugar transferase in the cis-Golgi cisterna recognizes newly synthesized lysosomal enzymes and adds a unique sugar. This enzyme adds N-acetylglucosamine phosphate to the mannose residues at the termini of the lysosomal enzymes' N-linked sugar trees. This enzyme differs from the usual sugar transferases (see p. 38) in that it adds a phosphosugar group to the mannose residue, rather than just a sugar. This enzyme is also unique in recognizing specific amino-acid sequences that are exclusively in these lysosomal enzymes. A second cis-Golgi enzyme removes the additional N-acetylglucosamine sugar, leaving its phosphate group behind. As a result, the sugar trees of the lysosomal enzymes terminate in mannose-6-phosphate residues (see Fig. 2-21, stage 2).

A special class of mannose-6-phosphate receptors, localized predominantly in the elements of the trans Golgi, recognize proteins that carry mannose-6-phosphate groups (see Fig. 2-21, stage 3). This recognition step constitutes the first stage of the cosegregation and clustering process discussed above. The mannose-6-phosphate receptors are transmembrane proteins. Their luminal portions bind to the newly synthesized lysosomal enzymes, whereas their cytoplasmically facing tails possess a particular signal that allows them to interact with adaptins and hence to be incorporated into clathrin-coated vesicles. The assembly of the clathrin lattice causes the mannose-6-phosphate receptors to cluster, along with their associated lysosomal enzymes, in the plane of the TGN membrane. Completion of the clathrin cage results in the formation of a vesicle whose membrane contains the mannose-6-phosphate receptors that bind their cargo of lysosomal enzymes.

After departing the TGN, these transport vesicles lose their clathrin coats (see Fig. 2-21, stage 4) and fuse with structures referred to as late endosomes or prelysosomal endosomes. Proton pumps in the membranes of these organelles ensure that their luminal pH is acidic (see Fig. 2-21, stage 5). When exposed to this acidic environment, the mannose-6-phosphate receptors undergo a conformational change that releases the mannose-6-phosphate–bearing lysosomal enzymes (see Fig. 2-21, stage 6). Consequently, the newly synthesized enzymes are dumped into the lumen of the prelysosomal endosome, which will go on to fuse with or mature into a lysosome. The empty mannose-6-phosphate receptors join vesicles that bud off from the lysosome (see Fig. 2-21, stage 7) and return to the TGN (see Fig. 2-21, stage 8). The luminal environment of the TGN allows the receptors to recover their affinity for mannose-6-phosphate, which permits them to participate in subsequent rounds of sorting.

Disruption of lysosomal sorting can be produced in several ways. For example, a drug called tunicamycin blocks the addition of N-linked sugars to newly synthesized proteins and thereby prevents attachment of the mannose-6-phosphate recognition marker. Compounds that elevate the luminal pH of the prelysosomal endosomes prevent newly synthesized enzymes from dissociating from the mannose-6-phosphate receptors and consequently block recycling of the receptor pool back to the TGN. The resulting shortage of unoccupied receptors allows mannose-6-phosphate–bearing proteins to pass through the TGN unrecognized (Box 2-2). Thus, instead of diverting to the lysosomes, these lysosomal enzymes continue along the secretory pathway and are ultimately released from the cell by constitutive secretion.

Box 2-2

Lysosomal Storage Diseases

In lysosomal storage diseases, the absence of a particular hydrolase—or group of hydrolases—from the lysosome prevents the lysosome from degrading certain substances, resulting in the formation of overstuffed lysosomes that crowd the cytoplasm and impede cell function. Indeed, a naturally occurring human disease—caused by a genetic defect in the sorting machinery—allowed the elucidation of lysosomal enzyme sorting.

In I-cell disease, most hydrolases are missing from the lysosomes of many cell types. As a result, lysosomes become engorged with massive quantities of undigested substrates. The enormously swollen lysosomes that characterize this disease were named inclusion bodies, and the cells that possess them were designated inclusion cells, or I cells for short. Whereas I cells lack most lysosomal enzymes, the genes that encode all of the hydrolases are completely normal. The mutation responsible for I-cell disease resides in the gene for the phosphosugar transferase that creates the mannose-6-phosphate recognition marker (see Fig. 2-21). Without this enzyme, the cell cannot sort any of the hydrolases to the lysosomes. Instead, the hydrolases pass through the TGN unnoticed by the mannose-6-phosphate receptors and are secreted constitutively from the affected cells.

In some other lysosomal storage diseases, specific hydrolases are not missorted but rather are genetically defective. For example, children who suffer from Tay-Sachs disease carry a homozygous mutation in the gene that encodes the lysosomal enzyme hexosaminidase A (HEX A). Consequently, their lysosomes are unable to degrade substances that contain certain specific sugar linkages. Because they cannot be broken down, these substances accumulate in lysosomes. Over time, these substances fill the lysosomes, which swell and crowd the cytoplasm. The resulting derangements of cellular function are toxic to a number of cell types and ultimately underlie this disease's uniform fatality within the first few years of life. Carriers of the Tay-Sachs trait can be detected either by HEX A enzyme testing or by DNA analysis of the HEX A gene. Among the Ashkenazi Jewish population, in whom 1 in 27 individuals is a carrier, three distinct HEX A mutations account for 98% of all carrier mutations.

Cells internalize extracellular material and plasma membrane through the process of endocytosis

The same fundamental mechanisms in the secretory pathway that produce vesicles by evaginating regions of Golgi membrane can also move material in the opposite direction by inducing vesicle formation through the invagination of regions of the plasma membrane. Vesicles created in this fashion are delimited by membrane that had formerly been part of the cell surface, and their luminal contents derive from the extracellular compartment.

This internalization process, referred to as endocytosis, serves the cell in at least four ways. First, certain nutrients are too large to be imported from the ECF into the cytoplasm via transmembrane carrier proteins; they are instead carried into the cell by endocytosis. Second, endocytosis of hormone-receptor complexes can terminate the signaling processes that are initiated by numerous hormones. Third, endocytosis is the first step in remodeling or degrading portions of the plasma membrane. Membrane that is delivered to the surface during exocytosis must be retrieved and ultimately returned to the TGN. Fourth, proteins or pathogens that need to be cleared from the extracellular compartment are brought into the cell by endocytosis and subsequently condemned to degradation in the lysosomes. Because endocytosed material can pursue a number of different destinies, there must be sorting mechanisms in the endocytic pathway, just as in the secretory pathway, that allow the cell to direct the endocytosed material to its appropriate destination.

Fluid-phase endocytosis is the uptake of the materials that are dissolved in the ECF (Fig. 2-22, stage 1) and not specifically bound to receptors on the cell surface. This process begins when a clathrin cage starts to assemble on the cytoplasmic surface of the plasma membrane. Above we discussed the physiology of clathrin-coated vesicles in the secretory pathway (see Fig. 2-19). The clathrin attaches to the membrane through interactions with adaptin proteins, which in turn adhere to the cytoplasmic tail domains of certain transmembrane polypeptides. Construction of the cage causes its adherent underlying membrane to invaginate and to form a coated pit (see Fig. 2-22, stage 2A). Completion of the cage creates a closed vesicle, which detaches from the cell surface through the process of membrane fission (see Fig. 2-22, stage 3). The resultant vesicle quickly loses its clathrin coat through the action of the uncoating ATPase and fuses with an organelle called an endosome.


FIGURE 2-22 Endocytosis.

Receptor-mediated endocytosis is responsible for internalizing specific proteins

Most of the proteins that a cell seeks to import via endocytosis are present in the ECF in extremely low concentrations. Furthermore, the volume of ECF that is internalized by an individual coated vesicle is very small. Consequently, the probability that any particular target molecule will enter the cell during a given round of fluid-phase endocytosis is low. To improve the efficiency of endocytosis and to ensure that the desired extracellular components are gathered in every endocytic cycle, the cell has devised a method for concentrating specific proteins at the site of endocytosis before initiating their uptake.

This concentration is achieved in a process known as receptor-mediated endocytosis, in which molecules to be internalized (see Fig. 2-22, stage 1) bind to cell-surface receptors with high affinity (see Fig. 2-22, stage 2B). Through this interaction, the substrates for endocytosis become physically associated with the plasma membrane, which greatly enhances the probability that they will be successfully internalized. Cells increase this probability even further by ensuring that the receptors themselves cluster in regions of the membrane destined to be endocytosed. The cytoplasmic tails of these receptors are endowed with recognition sequences that allow them to serve as binding sites for adaptins. Consequently, these receptors congregate in regions of the cell membrane where clathrin cages are assembling and are incorporated into coated pits as they are forming. The affinity of these receptors for the endocytic machinery ensures that their ligands are internalized with maximum efficiency.

Many endocytic receptors are constitutively associated with coated pits and are endocytosed whether or not they have bound their specific ligands. The cytoplasmic tails of certain receptors, however, interact with adaptins only when the receptor is in the bound state. For example, in the absence of epidermal growth factor (EGF), the EGF receptor is excluded from regions of the membrane in which coated pits are assembling. Modifications induced by ligand binding alter these receptors' tails, which allows them to participate in coated vesicle formation and hence in endocytosis.

After the clathrin-coated vesicle forms (see Fig. 2-22, stage 3), it quickly loses its clathrin coat, as described above for fluid-phase endocytosis, and fuses with an endosome. Although endosomes can be wildly pleomorphic, they frequently have an appearance like a frying pan, in which a round vesicular body is attached to a long tubular “handle” (see Fig. 2-22, stage 4). The cytoplasmic surfaces of the handles are often decorated with forming clathrin lattices and are the sites of vesicular budding.

Endocytosed proteins can be targeted to lysosomes or recycled to the cell surface

In many cell types, endocytosis is so rapid that, each hour, the cell internalizes a quantity of membrane that is equivalent in area to the entire cell surface. To persist in the face of this tremendous flux of membrane, the cell must retrieve most of the endocytosed membrane components and return them to the plasma membrane. However, substances that a cell wishes to degrade must be routed to lysosomes and prevented from escaping back to the surface. The sophisticated sorting operation required to satisfy both of these conditions takes place in the endosome.

Proton pumps embedded in its membrane ensure that, like the lysosome, the endosome maintains an acidic luminal pH (see Fig. 2-22, stage 4). This acidic environment initiates the separation of material that is destined for lysosomal destruction from those proteins that are to be recycled. Most endocytic receptors bind their ligands tightly at neutral pH but release them rapidly at pH values <6.0. Therefore, as soon as a surface-derived vesicle fuses with an endosome, proteins that are bound to receptors fall off and enter the endosomal lumen. The receptor proteins segregate in the membranes of the handles of the frying pan–shaped endosomes and are ultimately removed from the endosome in vesicles that shuttle them back to the cell surface (see Fig. 2-22, stage 5). The soluble proteins of the endosome lumen, which include the receptors' former ligands, are ultimately delivered to the lysosome. This sorting scheme allows the receptors to avoid the fate of their cargo and ensures that the receptors are used in many rounds of endocytosis.

The low-density lipoprotein (LDL) receptorimage N2-12 follows this regimen precisely. On arrival of the LDL-laden receptor at the endosome, the acidic environment of the endosome induces the LDL to dissociate from its receptor, which then promptly recycles to the cell surface. The LDL travels on to the lysosome, where enzymes destroy the LDL and liberate its bound cholesterol.


Familial Hypercholesterolemia

A Defect in Receptor-Mediated Endocytosis

Contributed by Michael Caplan

The critical physiologic role played by endocytic receptors is underscored by the molecular pathogenesis of a relatively common human genetic disease. Cholesterol is carried through the bloodstream bound to binding proteins, one of which is known as low-density lipoprotein, or LDL. Many cells throughout the body express receptors for LDL at their plasma membranes. These cells internalize receptor-bound LDL and utilize its cargo of cholesterol for membrane synthesis and in various biochemical pathways. The imported cholesterol also serves to inhibit the cells' endogenous de novo synthesis of cholesterol. The disease known as familial hypercholesterolemia (FHC) is caused by a defect in the gene encoding the LDL receptor, resulting in the synthesis of receptors that either do not bind LDL or fail to be internalized.

In the absence of functional LDL receptors, cells are unable to import exogenous cholesterol. Even though serum cholesterol levels rise to extraordinarily high levels, cells are oblivious to its presence, inasmuch as they lack the machinery that allows them to endocytose LDL. Consequently, their cholesterol synthesis continues uninhibited. The excess cholesterol synthesis results in the buildup of cholesterol-filled lipid droplets in cells throughout the body. Accumulation of these cholesterol inclusions in the cells lining arterial walls produces atherosclerotic plaques, which can go on to occupy and occlude the lumens of the blood vessels themselves. When these plaques appear in the coronary arteries, which supply the heart (see p. 560), they impede coronary blood flow and thus prevent the heart from receiving sufficient oxygen. Not surprisingly, patients with FHC often succumb to heart disease at relatively early ages. Patients who are homozygous for defective LDL receptors usually experience their first myocardial infarction (“heart attack”) in their early teens, whereas heterozygotes (whose cells express half the normal complement of functional LDL receptor) generally experience cardiac symptoms in midlife. The heterozygous form of FHC affects ~1 in 500 people. Our current understanding of receptor-mediated endocytosis, and of the molecular interactions on which it depends, owes a tremendous amount to insights gained through the study of FHC by Brown and Goldstein, whose work earned them the 1985 Nobel Prize in Medicine or Physiology. For more information about Brown and Goldstein and the work that led to their Nobel Prize, visit (accessed October 2014).

A variation on this paradigm is responsible for the cellular uptake of iron. Iron circulates in the plasma bound to a protein called transferrin. At the mildly alkaline pH of ECF, the iron-transferrin complex binds with high affinity to a transferrin receptor in the plasma membranes of almost every cell type. Bound transferrin is internalized by endocytosis and delivered to endosomes. Instead of inducing transferrin to fall off its receptor, the acid environment of the endosome lumen causes iron to fall off transferrin. Apotransferrin (i.e., transferrin without bound iron) remains tightly bound to the transferrin receptor at an acidic pH. The released iron is transported across the endosomal membrane for use in the cytosol. The complex of apotransferrin and the transferrin receptor recycles to the cell surface, where it is again exposed to the ECF. The mildly alkaline extracellular pH causes the transferrin receptor to lose its affinity for apotransferrin and to promptly release it. Thus, the cell uses the pH-dependent sorting trick twice to ensure that both the transferrin receptor and apotransferrin recycle for subsequent rounds of iron uptake.

Certain molecules are internalized through an alternative process that involves caveolae

Clathrin-coated pits are not the only cellular structures involved in receptor-mediated internalization. Electron microscopic examination of vascular endothelial cells that line blood vessels long ago revealed the presence of clusters of small vesicles that display a characteristic appearance, in close association with the plasma membrane. These caveolae were thought to be involved in the transfer of large molecules across the endothelial cells, from the blood space to the tissue compartment. In fact, caveolae are present in most cell types. The caveolae are rich in cholesterol and sphingomyelin. Rather than having a clathrin lattice, they contain intrinsic membrane proteins called caveolins, which face the cytosol (see Fig. 2-22). In addition, caveolae appear to be rich in membrane-associated polypeptides that participate in intracellular signaling, such as the Ras-like proteins (see p. 56) as well as heterotrimeric GTP-binding proteins (see p. 52). Caveolae appear to serve as organizing centers for collections of signaling molecules, perhaps allowing them to coordinate their activities. Caveolae also participate in the endocytosis of certain substances. In vascular endothelial cells, for example, caveolae take up albumin and carry it across the cell to the opposite side in a process known as transcytosis (see p. 467). Caveolae-mediated uptake does not involve clathrin or the acidified endocytic structures of the standard endocytotic pathway.

Also enriched in caveolae is the receptor for folate, a vitamin required by several metabolic pathways (see pp. 933–935). Unlike the receptors in the plasma membrane (see pp. 17–18), the folate receptor has no cytoplasmic tail that might allow it to associate with coated pits. Instead, it belongs to the GPI-linked class of proteins (see p. 17) that are anchored to the membrane through covalent attachment to phospholipid molecules. It appears that caveolae mediate the internalization of folate. In fact, a large number and variety of GPI-linked proteins are embedded in the outer leaflet of the caveolar membrane that faces its lumen.