Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Intracellular sorting of macromolecules and maintenance of cellular compartments
Transport from the Golgi apparatus to secretory vesicles and the cell surface

Normally, the Golgi apparatus continuously buds off transport vesicles destined to fuse immediately with Cell/30.html">The Plasma Membrane. The transmembrane Proteins and Lipids of these vesicles replenish The Cell membrane with new proteins and lipids, while the soluble proteins inside the vesicles are secreted into the extracellular space. In this way, the cell produces, for example, Proteoglycans and Extracellular matrix proteins (see Section 14.2).

While this constitutive secretion occurs in all cell types, specialized secretory Cells feature an additional secretory pathway in which soluble proteins and other substances first accumulate in secretory granules and are subsequently released in response to an extracellular signal (known as regulated, or stimulated, secretion; Fig. 8-76).

In this section, we will analyze The Role of the Golgi apparatus in these Two Types of secretion and compare the mechanisms involved in their execution. We will also examine how Viruses hijack the sorting machinery of the host cell and how Viral Particles can be useful in elucidating various Intracellular Transport pathways.

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Fig. 8-76. The regulated and constitutive secretory pathways diverge in the trans Golgi network. Many soluble proteins are continuously discharged from the cell via the constitutive secretory pathway, which is present in all cells. This same mechanism supplies the plasma membrane with newly synthesized lipids and transmembrane proteins. Specialized secretory cells also possess a regulated secretory mechanism, whereby specific proteins in the trans Golgi network are directed into secretory vesicles. Within these vesicles, the proteins are concentrated and stored until an extracellular signal triggers their secretion.

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8.9.1. Secretory vesicles bud off from the trans Golgi network [61]

In cells where secretion occurs in response to an extracellular signal, the secreted proteins are concentrated and stored in secretory vesicles (often called secretory granules because of their dark core). Upon receiving the appropriate signal, they are released via exocytosis. Secretory vesicles bud off from the trans Golgi network. It is believed that their formation requires clathrin and associated proteins that form a "coat," because part of The surface of the forming vesicles is typically covered with clathrin. This coat is shed shortly after the vesicle is fully formed (Fig. 8-77).

Like the lysosomal Hydrolases described in the previous section, proteins destined for secretory vesicles (often referred to as secretory proteins) must be selected and packaged into appropriate vesicles in the trans Golgi network. Apparently, this involves the selective aggregation of secretory proteins. Under an Electron microscope, the resulting aggregates appear as electron-dense material in the trans Golgi network. The "sorting signal" that directs a protein to such aggregates remains unknown, but it is likely a signal patch common to many secretory proteins. This Conclusion is supported by the finding that if the Gene encoding a secretory protein is transferred into a different type of secretory cell that does not normally synthesize that protein, the foreign protein will likewise be packaged into secretory vesicles.

It is unclear how aggregates containing secretory proteins are selected during The formation of secretory vesicles. Secretory vesicles have unique Membrane Proteins, some of which may serve as receptors (in the trans Golgi network) to bind the aggregated material destined for packaging. Note that secretory vesicles are larger than the transport vesicles that carry lysosomal hydrolases, and the aggregates they contain are too large for every molecule of the secreted protein to bind to a receptor in the vesicle membrane, as occurs during lysosomal enzyme transport (see Fig. 8-73). The capture of these aggregates by secretory granules is rather reminiscent of particle uptake by phagocytosis at the cell surface, which also occurs with the participation of clathrin-coated membranes.

Fig. 8-77. Electron micrograph of secretory vesicles budding from the trans Golgi network in Insulin-secreting pancreatic cells. Antibodies conjugated to colloidal gold particles (black dots) were used to localize clathrin molecules. Immature secretory vesicles (black arrows) containing proinsulin molecules are coated with clathrin. Once the vesicle is formed, the clathrin coat is rapidly shed and is absent in mature secretory vesicles (light arrows). (Micrograph kindly provided by Lelio Orci.)

After immature secretory vesicles bud off from the trans Golgi network, they lose their coat, and their contents become highly concentrated. This Condensation occurs abruptly and is presumably driven by the acidification of the vesicle lumen due to The activity of an ATP-dependent proton pump in its membrane. The aggregation of secreted proteins (or other components) and their subsequent condensation in secretory vesicles result in a 200-fold increase in the concentration of these proteins compared to the Golgi apparatus. This allows secretory vesicles to release large amounts of material on demand.

8.9.2. Secretory vesicle membrane components are recycled [62]

Many secretory cells, such as pancreatic acinar cells, are polarized, and exocytosis occurs exclusively at their apical surface. The apical region of the cells typically faces the lumen of a duct system that collects the secretion. When a secretory vesicle fuses with the plasma membrane, its contents are expelled from the cell via exocytosis, and its membrane becomes part of the plasma membrane (see Section 6.5.1). This would be expected to vastly increase the surface area of the plasma membrane. In reality, this expansion is very short-lived because membrane patches are removed from the surface by endocytosis (or recycled) at almost the same rate as they are added by exocytosis (Fig. 8-78). Clearly, this membrane retrieval returns secretory vesicle membrane proteins to the Golgi apparatus, where they can be reused. Recycling ensures a constant distribution of membrane components among various cellular compartments.

Fig. 8-78. After a secretory vesicle fuses with the plasma membrane at the apical pole of the cell, its membrane enters the recycling pathway. The amount of membrane added to the plasma membrane during regulated secretion can be massive. However, clathrin-coated vesicles are continuously formed from it and returned to the trans Golgi network (presumably via endosomes); this maintains a constant area of the cell's apical surface.

8.9.3. In unpolarized cells, proteins and lipids appear to be automatically transferred from the ER and Golgi apparatus to the cell surface [63]

In cells capable of regulated secretion, proteins must be sorted into at least three groups before leaving the trans Golgi network. The first group includes proteins destined for endolysosomes; the second, proteins destined for secretory vesicles; and finally, the third group comprises proteins that are delivered directly to the cell surface. Proteins heading to endolysosomes are selected for packaging into vesicles by a signal (M6P in the case of lysosomal hydrolases); similarly, every protein entering secretory vesicles must possess A special signal. Proteins of the third group are likely transported to the cell surface in a "non-selective" manner (Fig. 8-79). According to one hypothesis, in unpolarized cells (such as leukocytes and most cultured cells), any protein leaving the ER—unless it is retained as a permanent resident of that organelle or the Golgi apparatus, or selected for specific transport—is automatically carried to the cell surface. The appeal of this hypothesis lies in its simplicity and its ability to explain how damaged or misaddressed proteins are cleared from the cell.

An attempt was made to experimentally test the possibility of such a non-selective "bulk-flow" transport to the plasma membrane. To this end, cultured cells were incubated with a simple tripeptide (Asn-Tyr-Thr) containing the Asn-X-Thr glycosylation signal. This small peptide can penetrate cells and cross intracellular membranes; within the ER lumen, it becomes glycosylated at the asparagine residue. The addition of the N-linked oligosaccharide prevents the tripeptide from diffusing back into the Cytosol. Instead, it is transported in a one-way direction from the ER to the Golgi apparatus and then to the cell membrane. This process takes about 10 minutes, which matches the transport rate of the fastest plasma membrane proteins. This result is consistent with the hypothesis that a non-selective flow of fluid filling the lumen of Organelles exists, which automatically delivers any soluble molecule from the ER to the cell surface (unless it is retained within an organelle or directed elsewhere by a signal).

Fig. 8-79. The best-characterized protein sorting pathways in the trans Golgi network. Proteins bearing a mannose 6-phosphate marker are directed to Lysosomes (via endolysosomes) within clathrin-coated vesicles (see Fig. 8-72). Proteins destined for secretion are concentrated in large clathrin-coated vesicles that lose their coats to become secretory vesicles—a pathway found exclusively in specialized secretory cells. It is thought that in unpolarized cells, proteins lacking special signals are directed to the cell surface "by default" via constitutive secretion. In polarized cells, secreted proteins and plasma membrane proteins are sorted selectively to either the apical or basolateral domain of the plasma membrane, and therefore at least one of these pathways must be regulated by specific signals.

In principle, this same non-selective flow can deliver transmembrane proteins and lipids that lack sorting signals to the cell surface. It can also transport proteins destined for secretory vesicles from the ER to the distal side of the Golgi apparatus, since specific signals distinguishing these proteins from those heading to the plasma membrane are required only in the trans Golgi network. A special sorting mechanism is likely required for a protein to remain in the ER or Golgi apparatus (see Section 8.1.5). Recent data showing that certain resident ER proteins (including BiP and protein disulfide isomerase) contain a signal peptide responsible for their ER localization (see Section 8.1.7) support this general view of Intracellular Protein Transport.

Some constitutively secreted proteins take a long time to leave the ER and be secreted. To reconcile these findings with the bulk-flow hypothesis, it has been suggested that such proteins require time to fold properly and are therefore retained in the ER for an extended period, either by spanning the ER membrane or by binding to special proteins such as BiP. Once properly folded, these proteins also enter the bulk-flow pathway.

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8.9.4. Polarized cells can direct proteins from the Golgi apparatus to a specific region of the plasma membrane [64]

Most cells in Tissues are polarized, and their plasma membrane consists of two (or sometimes more) distinct domains or regions. For example, a typical epithelial cell has two physically continuous yet biochemically distinct Regions of the cell membrane (see Fig. 6-36): the apical region faces the lumen of an organ and often bears specialized adaptations, such as cilia or a microvillar brush border; the basolateral region covers the rest of the cell. These two regions are joined at their boundary by a ring of tight junctions (see Section 14.1.1) that prevent proteins (and lipids in the outer leaflet of The Lipid Bilayer) from diffusing from one membrane region to the other. This is why, although both membrane regions appear continuous under an electron microscope, they are effectively isolated from each other by tight junctions and contain different sets of proteins. The Lipid Composition of the two bilayers also differs; in particular, Glycolipids are found exclusively in the apical membrane region. There is compelling evidence showing that the sets of proteins secreted from the apical and basolateral surfaces of an epithelial cell are likewise distinct. Consequently, polarized cells must possess mechanisms that specifically direct both membrane and secretory proteins to a particular domain of the plasma membrane. Cultured cell studies have established that proteins destined for different domains travel together from the ER to the trans Golgi network, where they are sorted and packaged into secretory or transport vesicles destined for the appropriate regions of the cell membrane. It is possible that both apical and basolateral regions bear sorting signals that direct proteins to the corresponding domain; alternatively, only one of these pathways may require a specific signal, with the other operating by default in its absence.

Fig. 8-80. Structure of Semliki Forest virus. Schematic cross-section of the virus (A) and three-dimensional reconstruction of its surface obtained from cryo-Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF unstained preparations (B). Each viral particle contains 180 copies of the capsid protein C (assembled into 60 trimers) and 240 copies of each of the three envelope proteins (assembled into 80 trimers). The outer viral envelope consists of envelope proteins embedded in a lipid bilayer. The virus has a total molecular mass of 46 million daltons. (B, courtesy of S. Fuller, Cell, 48: 923-934, 1987).

8.9.5. Viruses hijack the sorting machinery of the host cell [65]

The Genome of many animal viruses consists of a small amount of nucleic acid and comprises no more than four or five genes. Most of these genes encode structural proteins of the mature viral particle (virion), so viruses must rely on the appropriate host cell machinery during Replication. Because viral products are typically produced in large quantities during an infection, and viral particles sequentially traverse various host cell compartments throughout their life cycle, virus-infected cells serve as an exceptionally useful model for studying intracellular transport pathways.

Enveloped animal viruses, whose genome is enclosed in a Lipid Bilayer Membrane (see Section 5.5.2), exploit cellular compartmentalization with remarkable efficiency. Tracing The life cycle of such a virus is akin to taking a journey through the cell. A well-studied example is the Semliki Forest virus, whose genome consists of RNA surrounded by a capsid composed of a regular icosahedral (20-sided) protein shell. The capsid protein is designated as the C protein. The nucleocapsid (genome + capsid) is surrounded by a tightly fitting lipid bilayer containing only three proteins (designated E1, E2, and E3). These proteins (envelope proteins) are Glycoproteins that span the lipid bilayer and link the membrane and nucleocapsid together (Fig. 8-80, A). The glycosylated portions of the envelope proteins always project outward from the lipid bilayer, and complexes of these proteins form "spikes" On the surface of the viral particle that can be visualized by Electron Microscopy (Fig. 8-80, B).

Infection begins when the virus binds to a receptor protein on the plasma membrane of the host cell. To enter the cell, the virus hijacks the normal receptor-mediated endocytosis pathway, thereby gaining entry into endosomes (see Section 6.5.8). However, rather than being subsequently delivered to lysosomes, the virus escapes the endosomes due to the unique properties of one of its envelope proteins. At the acidic pH characteristic of endosomes, this protein triggers the fusion of the viral envelope with the endosomal membrane, releasing the nucleocapsid into the cytosol (Fig. 8-81). In the cytosol, the nucleocapsid "uncoats" to release the viral RNA, which is then translated by host cell Ribosomes to produce virus-encoded RNA polymerase. This enzyme, in turn, synthesizes many copies of the RNA. Some of these copies subsequently serve as mRNAs, directing the Synthesis of the four structural viral proteins: the capsid C protein and the three envelope proteins E1, E2, and E3.

Envelope and capsid proteins follow distinct intracellular pathways. Envelope proteins, like normal cellular glycoproteins, are synthesized on ER-bound ribosomes; the capsid protein, acting like a typical cytosolic protein, is synthesized on free ribosomes. Newly synthesized capsid protein binds to newly replicated viral RNA to form a new nucleocapsid. By contrast, envelope proteins are inserted into the ER membrane, where they are glycosylated, transported to the Golgi apparatus (where their Oligosaccharides are modified), and finally delivered to the plasma membrane.

Fig. 8-81. Life Cycle of Semliki Forest virus. During most stages of Biosynthesis, this virus parasitizes the host cell.

Ultimately, viral nucleocapsids and envelope proteins "meet" at the plasma membrane (see Fig. 8-81). Through a specific interaction with a cluster of envelope proteins, the nucleocapsid becomes wrapped in the plasma membrane to form a "bud," the membrane of which is heavily enriched in viral envelope proteins while retaining host cell lipids. Finally, this bud pinches off, releasing the free viral particle from the cell. The clustering of envelope proteins in the lipid bilayer can be viewed as a model for the segregation of specific membrane proteins during the formation of coated vesicles.

8.9.6. Viral envelope proteins carry signals that direct them to specific intracellular membranes [66]

Enveloped viral particles bud from various regions of the host cell membrane. Consequently, their envelope proteins (which are transmembrane proteins synthesized in the ER) must carry signals that direct them from the ER to a specific cellular membrane. When epithelial cells are cultured on a substrate of Collagen-coated porous filters, they form polarized cell sheets. If such polarized cells are infected with a virus (while maintaining the distinction between apical and basolateral membrane domains), some viruses (such as Influenza virus) bud exclusively from the apical plasma membrane, whereas others (such as Semliki Forest virus and vesicular stomatitis virus) bud exclusively from the basolateral domain (Fig. 8-82). This polarity reflects the presence of distinct apical or basolateral sorting signals on the viral envelope proteins, which direct these proteins to only one domain of the cell surface; in turn, the envelope proteins drive virus assembly at that specific domain.

Fig. 8-82. Electron micrographs showing that one type of enveloped virus buds from the apical surface and another from the basolateral surface of the same cultured epithelial cells. These cells grow with their basal surface attached to the culture dish. (Courtesy of E. Rodriguez-Boulan and D. Sabatini.)

The envelope proteins of other viruses carry different sorting signals. For example, herpesvirus is a DNA-containing virus that replicates in the Cell Nucleus, where its nucleocapsid is also assembled. The virus subsequently acquires an envelope by budding from the inner nuclear membrane into the ER lumen. Thus, its envelope proteins must be transported from the ER membrane to the inner nuclear membrane. By contrast, Flaviviruses bud directly into the ER lumen, whereas Bunyaviruses bud into the Golgi apparatus; this means their envelope proteins carry signals that retain them in the ER and Golgi membranes, respectively. Particles of herpesvirus, flavivirus, and bunyavirus accumulate within the lumen of the ER and Golgi apparatus and move toward the cell surface precisely as if they were secretory proteins; in the trans Golgi network, they are incorporated into transport vesicles and dispatched from the cell via the constitutive secretory pathway.

Conclusion

Proteins can be exported from the cell via exocytosis through either the constitutive or the regulated pathway. In the regulated pathway, molecules are stored in secretory vesicles that do not fuse with the plasma membrane or release their contents until an extracellular signal is received. The packaging of proteins into these vesicles in the trans Golgi network is accompanied by their selective condensation. Regulated secretion occurs exclusively in specialized secretory cells, whereas the constitutive secretory mechanism is present in all cells. The primary route of constitutive secretion is vesicular transport from the trans Golgi network to the plasma membrane. In unpolarized cells, proteins that are not specifically targeted to a particular organelle and lack retention sorting signals are automatically delivered by the constitutive pathway to the trans Golgi network and thence to the plasma membrane. In polarized cells, transport pathways from the trans Golgi network to the plasma membrane must operate selectively to ensure the targeted delivery of distinct sets of membrane proteins, secretory proteins, and lipids to the apical and basolateral membrane domains.