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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.
As discussed in Chapter 12, newly synthesized proteins enter the biosynthetic- secretory pathway in the ER by crossing the ER membrane from the cytosol. During their subsequent transport, from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface and elsewhere, these proteins pass through a series of compartments, where they are successively modified. Transfer from one compartment to the next involves a delicate balance between forward and backward (retrieval) transport pathways. Some transport vesicles select cargo molecules and move them to the next compartment in the pathway, while others retrieve escaped proteins and return them to a previous compartment where they normally function. Thus, the pathway from the ER to the cell surface involves many sorting steps, which continually select membrane and soluble lumenal proteins for packaging and transport—in vesicles or organelle fragments that bud from the ER and Golgi apparatus.
In this section we focus mainly on the Golgi apparatus (also called the Golgi complex). It is a major site of carbohydrate synthesis, as well as a sorting and dispatching station for the products of the ER. Many of the cell's polysaccharides are made in the Golgi apparatus, including the pectin and hemicellulose of the cell wall in plants and most of the glycosaminoglycans of the extracellular matrix in animals (discussed in Chapter 19). But the Golgi apparatus also lies on the exit route from the ER, and a large proportion of the carbohydrates that it makes are attached as oligosaccharide side chains to the many proteins and lipids that the ER sends to it. A subset of these oligosaccharide groups serve as tags to direct specific proteins into vesicles that then transport them to lysosomes. But most proteins and lipids, once they have acquired their appropriate oligosaccharides in the Golgi apparatus, are recognized in other ways for targeting into the transport vesicles going to other destinations.
To initiate their journey along the biosynthetic-secretory pathway, proteins that have entered the ER and are destined for the Golgi apparatus or beyond are first packaged into small COPII-coated transport vesicles. These transport vesicles bud from specialized regions of the ER called ER exit sites, whose membrane lacks bound ribosomes. In most animal cells, ER exit sites seem to be randomly dispersed throughout the ER network.
Originally it was thought that all proteins that are not tethered in the ER enter transport vesicles by default. However, it is now clear that packaging into vesicles that leave the ER can also be a selective process. Some cargo proteins are actively recruited into such vesicles, where they become concentrated. It is thought that these cargo proteins display exit (transport) signals on their surface that are recognized by complementary receptor proteins that become trapped in the budding vesicle by interacting with components of the COPII coat (Figure 13-17). At a much lower rate, proteins without such exit signals can also get packaged in vesicles, so that even proteins that normally function in the ER (so-called ER resident proteins) slowly leak out of the ER. Similarly, secretory proteins that are made in high concentrations may leave the ER without the help of sorting receptors.
The recruitment of cargo molecules into ER transport vesicles. By binding to the COPII coat, membrane and cargo proteins become concentrated in the transport vesicles as they leave the ER. Membrane proteins are packaged into budding transport vesicles (more...)
The exit signals that direct proteins out of the ER for transport to the Golgi and beyond are mostly not understood. There is one exception, however. The ERGIC53 protein seems to serve as a receptor for packaging some secretory proteins into COPII-coated vesicles. Its role in protein transport was identified because humans who lack it owing to an inherited mutation have lowered serum levels of two secreted blood-clotting factors (Factor V and Factor VIII) and therefore bleed excessively. The ERGIC53 protein is a lectin that binds mannose and is thought to recognize this sugar on Factor V and Factor VIII proteins, thereby packaging the proteins into transport vesicles in the ER.
To exit from the ER, proteins must be properly folded and, if they are subunits of multimeric protein complexes, they may need to be completely assembled. Those that are misfolded or incompletely assembled are retained in the ER, where they are bound to chaperone proteins (see Chapter 6), such as BiP or calnexin. The chaperones may cover up the exit signals or somehow anchor the proteins in the ER (Figure 13-18). Such failed proteins are eventually transported back into the cytosol where they are degraded by proteasomes (discussed in Chapter 12). This quality-control step is important, as misfolded or misassembled proteins could potentially interfere with the functions of normal proteins if they were transported onward. The amount of corrective action is surprisingly large. More than 90% of the newly synthesized subunits of the T cell receptor (discussed in Chapter 24) and of the acetylcholine receptor (discussed in Chapter 11), for example, are normally degraded in the cell without ever reaching the cell surface, where they function. Thus, cells must make a large excess of many protein molecules from which to select the few that fold and assemble properly.
Retention of incompletely assembled antibody molecules in the ER. Antibodies are made up of two heavy and two light chains (discussed in Chapter 24), which assemble in the ER. The chaperone BiP is thought to bind to all incompletely assembled antibody (more...)
Sometimes, however, this quality-control mechanism is detrimental. The predominant mutations that cause cystic fibrosis, a common inherited disease, produce a plasma membrane protein important for Cl- transport that is only slightly misfolded. Although the mutant protein would function perfectly normally if it reached the plasma membrane, it is retained in the ER. The devastating disease thus results not because the mutation inactivates the protein, but because the active protein is discarded before it reaches the plasma membrane.
After transport vesicles have budded from an ER exit site and have shed their coat, they begin to fuse with one another. This fusion of membranes from the same compartment is called homotypic fusion, to distinguish it from heterotypic fusion, in which a membrane from one compartment fuses with the membrane of a different compartment. As with heterotypic fusion, homotypic fusion requires a set of matching SNAREs. In this case, however, the interaction is symmetrical, with v-SNAREs and t-SNAREs contributed by both membranes (Figure 13-19).
Homotypic membrane fusion. In step 1, identical pairs of v-SNAREs and t-SNAREs in both membranes are pried apart by NSF (see Figure 13-13). In steps 2 and 3, the separated matching SNAREs on adjacent identical membranes interact, which leads to membrane (more...)
The structures formed when ER-derived vesicles fuse with one another are called vesicular tubular clusters, on the basis of their convoluted appearance in the electron microscope (Figure 13-20A). These clusters constitute a new compartment that is separate from the ER and lacks many of the proteins that function in the ER. They are generated continually and function as transport packages that bring material from the ER to the Golgi apparatus. The clusters are relatively short-lived because they quickly move along microtubules to the Golgi apparatus, where they fuse and deliver their contents (Figure 13-20B).
Vesicular tubular clusters. (A) An electron micrograph section of vesicular tubular clusters forming from the ER membrane. Many of the vesicle-like structures seen in the micrograph are cross sections of tubules that extend above and below the plane of (more...)
As soon as vesicular tubular clusters form, they begin budding off vesicles of their own. Unlike the COPII-coated vesicles that bud from the ER, these vesicles are COPI-coated. They carry back to the ER resident proteins that have escaped, as well as proteins that participated in the ER budding reaction and are being returned. This retrieval process demonstrates the exquisite control mechanisms that regulate coat assembly reactions. The COPI coat assembly begins only seconds after the COPII coats have been shed. It remains a mystery how this switchover in coat assembly is controlled.
The retrieval (or retrograde) transport continues as the vesicular tubular clusters move to the Golgi apparatus. Thus, the clusters continuously mature, gradually changing their composition as selected proteins are returned to the ER. A similar retrieval process continues from the Golgi apparatus, after the vesicular tubular clusters have delivered their cargo.
The retrieval pathway for returning escaped proteins back to the ER depends on ER retrieval signals. Resident ER membrane proteins, for example, contain signals that bind directly to COPI coats and are thus packaged into COPI-coated transport vesicles for retrograde delivery to the ER. The best-characterized signal of this type consists of two lysines, followed by any two other amino acids, at the extreme C-terminal end of the ER membrane protein. It is called a KKXX sequence, based on the single-letter amino acid code.
Soluble ER resident proteins, such as BiP, also contain a short retrieval signal at their C-terminal end, but it is different: it consists of a Lys-Asp-Glu-Leu or similar sequence. If this signal (called the KDEL sequence) is removed from BiP by genetic engineering, the protein is slowly secreted from the cell. If the signal is transferred to a protein that is normally secreted, the protein is now efficiently returned to the ER, where it accumulates.
Unlike the retrieval signals on ER membrane proteins that can interact directly with the COPI coat, soluble ER resident proteins must bind to specialized receptor proteins such as the KDEL receptor—a multipass transmembrane protein that binds to the KDEL sequence and packages any protein displaying it into COPI-coated retrograde transport vesicles. To accomplish this task, the KDEL receptor itself must cycle between the ER and the Golgi apparatus, and its affinity for the KDEL sequence must be different in these two compartments. The receptor must have a high affinity for the KDEL sequence in vesicular tubular clusters and the Golgi apparatus, so as to capture escaped ER resident proteins that are present there at low concentration. It must have a low affinity for the KDEL sequence in the ER, however, to unload its cargo in spite of the very high concentration of KDEL-containing resident proteins in the ER.
How can the affinity of the KDEL receptor change depending on the compartment in which it resides? The answer may be related to the different pH values established in the different compartments, regulated by H+ pumps in their membrane. The KDEL receptor could bind the KDEL sequence under the slightly acidic conditions in vesicular tubular clusters and the Golgi compartment but release it at the neutral pH in the ER. As we discuss later, such pH-sensitive protein-protein interactions form the basis for many of the sorting steps in the cell (Figure 13-21).
A model for the retrieval of ER resident proteins. Those ER resident proteins that escape from the ER are returned to the ER by vesicular transport. (A) The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus, captures the soluble (more...)
Most membrane proteins that function at the interface between the ER and Golgi apparatus, including v- and t-SNARES and some cargo receptors, enter the retrieval pathway to the ER. Whereas the recycling of some of these proteins is signal-mediated as just described, for others no specific signal seems to be required. Thus, while retrieval signals increase the efficiency of the retrieval process, some proteins—including some Golgi enzymes—randomly enter budding vesicles destined for the ER and are returned to the ER at a slower rate. Such Golgi enzymes cycle constantly between the ER and the Golgi, but their rate of return to the ER is slow enough for most of the protein to be found in the Golgi apparatus.
The KDEL retrieval pathway only partly explains how ER resident proteins are maintained in the ER. As expected, cells that express genetically modified ER resident proteins, from which the KDEL sequence has been experimentally removed, secrete these proteins. But secretion occurs at a much slower rate than for a normal secretory protein. It seems that ER resident proteins are anchored in the ER by a mechanism that is independent of their KDEL signal and that only those proteins that escape retention are captured and returned via the KDEL receptor. A suggested mechanism of retention is that ER resident proteins bind to one another, thus forming complexes that are too big to enter transport vesicles. Because ER resident proteins are present in the ER at very high concentrations (estimated to be millimolar), relatively low-affinity interactions would suffice to have most of the proteins tied up in such complexes.
Aggregation of proteins that function in the same compartment—called kin recognition—is a general mechanism that compartments use to organize and retain their resident proteins. Golgi enzymes that function together, for example, also bind to each other and are thereby restrained from entering transport vesicles.
Vesicles that leave the Golgi apparatus of animal cells destined for the plasma membrane are rich in cholesterol. The cholesterol fills the space between the kinked hydrocarbon chains of the lipids in the bilayer, forcing them into tighter alignment and increasing the separation between the lipid head groups of the two leaflets of the bilayer (see Figure 10-11). Thus the lipid bilayer of the cholesterol-derived vesicles is thicker than that of the Golgi membrane itself. Transmembrane proteins must have sufficiently long transmembrane segments to span this thickness if they are to enter the cholesterol-rich transport vesicle budding from the Golgi apparatus destined for the plasma membrane. Proteins with shorter transmembrane segments are excluded.
This exclusion is thought to explain why membrane proteins that normally reside in the Golgi and the ER have shorter transmembrane segments (around 15 amino acids) than do plasma membrane proteins (around 20–25 amino acids). When the transmembrane segments of Golgi proteins are extended by recombinant DNA techniques, the proteins are no longer efficiently retained in the Golgi apparatus and are transported to the plasma membrane instead. Thus, at least some Golgi proteins seem to be retained in the Golgi apparatus mainly because they cannot enter transport vesicles heading for the plasma membrane.
Because of its large and regular structure, the Golgi apparatus was one of the first organelles described by early light microscopists. It consists of a collection of flattened, membrane-enclosed cisternae, somewhat resembling a stack of pancakes. Each of these Golgi stacks usually consists of four to six cisternae (Figure 13-22), although some unicellular flagellates can have up to 60. In animal cells, many stacks are linked by tubular connections between corresponding cisternae, thus forming a single complex, which is usually located near the cell nucleus and close to the centrosome (Figure 13-23A). This localization depends on microtubules. If microtubules are experimentally depolymerized, the Golgi apparatus reorganizes into individual stacks that are found throughout the cytoplasm, adjacent to ER exit sites. In some cells, including most plant cells, hundreds of individual Golgi stacks are normally dispersed throughout the cytoplasm (Figure 13-23B).
The Golgi apparatus. (A) Three-dimensional reconstruction from electron micrographs of the Golgi apparatus in a secretory animal cell. The cis-face of the Golgi stack is that closest to the ER. (B) A thin-section electron micrograph emphasizing the transitional (more...)
Light micrographs of the Golgi apparatus. (A) The Golgi apparatus in a cultured fibroblast stained with a fluorescent antibody that recognizes a Golgi resident protein. The Golgi apparatus is polarized, facing the direction in which the cell was crawling (more...)
During their passage through the Golgi apparatus, transported molecules undergo an ordered series of covalent modifications. Each Golgi stack has two distinct faces: a cis face (or entry face) and a trans face (or exit face). Both cis and trans faces are closely associated with special compartments, each composed of a network of interconnected tubular and cisternal structures: the cis Golgi network (CGN) (also called the intermediate compartment) and the trans Golgi network (TGN), respectively. Proteins and lipids enter the cis Golgi network in vesicular tubular clusters arriving from the ER and exit from the trans Golgi network bound for the cell surface or another compartment. Both networks are thought to be important for protein sorting. As we have seen, proteins entering the CGN can either move onward in the Golgi apparatus or be returned to the ER. Similarly, proteins exiting from the TGN can either move onward and be sorted according to whether they are destined for lysosomes, secretory vesicles, or the cell surface, or be returned to an earlier compartment.
The Golgi apparatus is especially prominent in cells that are specialized for secretion, such as the goblet cells of the intestinal epithelium, which secrete large amounts of polysaccharide-rich mucus into the gut (Figure 13-24). In such cells, unusually large vesicles are found on the trans side of the Golgi apparatus, which faces the plasma membrane domain where secretion occurs.
A goblet cell of the small intestine. This cell is specialized for secreting mucus, a mixture of glycoproteins and proteoglycans synthesized in the ER and Golgi apparatus. Like all epithelial cells, goblet cells are highly polarized, with the apical domain (more...)
As described in Chapter 12, a single species of N -linked oligosaccharide is attached en bloc to many proteins in the ER and then trimmed while the protein is still in the ER. Further modifications and additions occur in the Golgi apparatus, depending on the protein. The outcome is that two broad classes of N-linked oligosaccharides, the complex oligosaccharides and the high-mannose oligosaccharides, are found attached to mammalian glycoproteins (Figure 13-25). Sometimes both types are attached (in different places) to the same polypeptide chain.
The two main classes of asparagine-linked (N-linked) oligosaccharides found in mature glycoproteins. (A) Both complex oligosaccharides and high-mannose oligosaccharides share a common core region derived from the original N-linked oligosaccharide added (more...)
Complex oligosaccharides are generated by a combination of trimming the original N-linked oligosaccharide added in the ER and the addition of further sugars. By contrast, high-mannose oligosaccharides have no new sugars added to them in the Golgi apparatus. They contain just two N-acetylglucosamines and many mannose residues, often approaching the number originally present in the lipid-linked oligosaccharide precursor added in the ER. Complex oligosaccharides can contain more than the original two N-acetylglucosamines as well as a variable number of galactose and sialic acid residues and, in some cases, fucose. Sialic acid is of special importance because it is the only sugar in glycoproteins that bears a negative charge. Whether a given oligosaccharide remains high-mannose or is processed is determined largely by its position on the protein. If the oligosaccharide is accessible to the processing enzymes in the Golgi apparatus, it is likely to be converted to a complex form; if it is inaccessible because its sugars are tightly held to the protein's surface, it is likely to remain in a high-mannose form. The processing that generates complex oligosaccharide chains follows the highly ordered pathway shown in Figure 13-26.
Oligosaccharide processing in the ER and the Golgi apparatus. The processing pathway is highly ordered, so that each step shown is dependent on the previous one. Processing begins in the ER with the removal of the glucoses from the oligosaccharide initially (more...)
It is not only the N-linked oligosaccharide chains on proteins that are altered as the proteins pass through the Golgi cisternae en route from the ER to their final destinations; many proteins are also modified in other ways. Some proteins have sugars added to the OH groups of selected serine or threonine side chains. This O -linked glycosylation, like the extension of N-linked oligosaccharide chains, is catalyzed by a series of glycosyl transferase enzymes that use the sugar nucleotides in the lumen of the Golgi apparatus to add sugar residues to a protein one at a time. Usually, N-acetylgalactosamine is added first, followed by a variable number of additional sugar residues, ranging from just a few to 10 or more.
The Golgi apparatus confers the heaviest glycosylation of all on proteoglycan core proteins, which it modifies to produce proteoglycans. As discussed in Chapter 19, this process involves the polymerization of one or more glycosaminoglycan chains (long unbranched polymers composed of repeating disaccharide units) via a xylose link onto serines on the core protein. Many proteoglycans are secreted and become components of the extracellular matrix, while others remain anchored to the plasma membrane. Still others form a major component of slimy materials, such as the mucus that is secreted to form a protective coating over many epithelia.
The sugars incorporated into glycosaminoglycans are heavily sulfated in the Golgi apparatus immediately after these polymers are made, thus adding a significant portion of their characteristically large negative charge. Some tyrosine residues in proteins also become sulfated shortly before they exit from the Golgi apparatus. In both cases, the sulfation depends on the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate, or PAPS, that is transported from the cytosol into the lumen of the trans Golgi network.
There is an important difference between the construction of an oligosaccharide and the synthesis of other macromolecules such as DNA, RNA, and protein. Whereas nucleic acids and proteins are copied from a template in a repeated series of identical steps using the same enzyme or set of enzymes, complex carbohydrates require a different enzyme at each step, each product being recognized as the exclusive substrate for the next enzyme in the series. Given the complicated pathways that have evolved to synthesize them, it seems likely that the oligosaccharides on glycoproteins and glycosphingolipids have important functions, but for the most part these functions are not known.
N-linked glycosylation, for example, is prevalent in all eucaryotes, including yeasts, but is absent from procaryotes. Because one or more N-linked oligosaccharides are present on most proteins transported through the ER and Golgi apparatus—a pathway that is unique to eucaryotic cells—one might suspect that they function to aid folding and the transport process. We have already discussed a number of instances for which this is so—the use of a carbohydrate as a marker during protein folding in the ER (see Chapter 12), for example, and the use of carbohydrate-binding lectins in guiding ER-to-Golgi transport. As we discuss later, lectins also participate in protein sorting in the trans Golgi network.
Because chains of sugars have limited flexibility, even a small N-linked oligosaccharide protrudes from the surface of a glycoprotein (Figure 13-27) and can thus limit the approach of other macromolecules to the protein surface. In this way, for example, the presence of oligosaccharides tends to make a glycoprotein more resistant to digestion by proteases. It may be that the oligosaccharides on cell-surface proteins originally provided an ancestral eucaryotic cell with a protective coat that, unlike the rigid bacterial cell wall, left the cell with the freedom to change shape and move. But if so, these sugar chains have since become modified to serve other purposes as well. The oligosaccharides attached to some cell-surface proteins, for example, are recognized by transmembrane lectins called selectins, which function in cell-cell adhesion processes, as discussed in Chapter 19.
The three-dimensional structure of a small N-linked oligosaccharide. The structure was determined by x-ray crystallographic analysis of a glycoprotein. This oligosaccharide contains only 6 sugar residues, whereas there are 14 sugar residues in the N-linked (more...)
Glycosylation can also have important regulatory roles. Signaling through the cell-surface signaling receptor Notch, for example, is important for proper cell fate determination in development. Notch is a transmembrane protein that is O-glycosylated by addition of a single fucose to some serines, threonines, and hydroxylysines. Some cell types express an additional glycosyltransferase that adds an N-acetylglucosamine to each of these fucoses in the Golgi apparatus. This addition sensitizes the Notch receptor, and thus allows these cells to respond selectively to activating stimuli. In this way, glycosylation has become important to the establishment of spatial boundaries in developing tissues.
Proteins exported from the ER enter the first of the Golgi processing compartments (the cis Golgi compartment), after having passed through the cis Golgi network. They then move to the next compartment (the medial compartment, consisting of the central cisternae of the stack) and finally to the trans compartment, where glycosylation is completed. The lumen of the trans compartment is thought to be continuous with the trans Golgi network, where proteins are segregated into different transport packages and dispatched to their final destinations—the plasma membrane, lysosomes, or secretory vesicles.
The oligosaccharide processing steps occur in a correspondingly organized sequence in the Golgi stack, with each cisterna containing a characteristic abundance of processing enzymes. Proteins are modified in successive stages as they move from cisterna to cisterna across the stack, so that the stack forms a multistage processing unit. This compartmentalization might seem unnecessary, since each oligosaccharide processing enzyme can accept a glycoprotein as a substrate only after it has been properly processed by the preceding enzyme. Nonetheless, it is clear that processing occurs in a spatial as well as a biochemical sequence: enzymes catalyzing early processing steps are concentrated in the cisternae toward the cis face of the Golgi stack, whereas enzymes catalyzing later processing steps are concentrated in the cisternae toward the trans face.
The functional differences between the cis, medial, and trans subdivisions of the Golgi apparatus were discovered by localizing the enzymes involved in processing N-linked oligosaccharides in distinct regions of the organelle, both by physical fractionation of the organelle and by labeling the enzymes in electron microscope sections with antibodies. The removal of mannose residues and the addition of N-acetylglucosamine, for example, were shown to occur in the medial compartment, while the addition of galactose and sialic acid was found to occur in the trans compartment and the trans Golgi network (Figure 13-28). The functional compartmentalization of the Golgi apparatus is summarized in diagrammatic form in Figure 13-29.
Histochemical stains demonstrating the biochemical compartmentalization of the Golgi apparatus. A series of electron micrographs shows the Golgi apparatus (A) unstained, (B) stained with osmium, which is preferentially reduced by the cisternae of the (more...)
The functional compartmentalization of the Golgi apparatus. The localization of each processing step shown was determined by a combination of techniques, including biochemical subfractionation of the Golgi apparatus membranes and electron microscopy after (more...)
The functional and structural divisions of the Golgi stack pose two important questions. How are molecules transported from one Golgi cisterna to the next, and how are Golgi resident proteins retained in their appropriate places?
It is still uncertain how the Golgi apparatus achieves and maintains its polarized structure and how molecules move from one cisterna to another. Functional evidence from in vitro transport assays and the finding of abundant transport vesicles in the vicinity of Golgi cisternae initially led to the view that these vesicles transport proteins between the cisternae, budding from one cisterna and fusing with the next. According to this vesicular transport model, the Golgi apparatus is a relatively static structure, with its enzymes held in place, while the molecules in transit are moved through the cisternae in sequence, carried by transport vesicles (Figure 13-30A). Retrograde flow retrieves escaped ER and Golgi proteins and returns them to preceding compartments. Directional flow is achieved as forward-moving cargo molecules are selectively packaged into forward-moving vesicles, whereas proteins to be retrieved are selectively packaged into retrograde vesicles. Although both types of vesicles are likely to be COPI-coated, the coats may contain different adaptor proteins to confer selectivity on the packaging of cargo molecules. Alternatively, transport vesicles that shuttle between Golgi cisternae may not be directional at all, transporting cargo material randomly back and forth; directional flow would then occur because of the continual input at the cis cisterna and output at the trans cisterna. In either case, the movement of vesicles from each cisterna to an adjacent one is helped by a neat trick: the budding vesicles remain tethered by filamentous proteins that restrict their movement, so that their fusion with the correct target membrane is facilitated.
Two possible models explaining the organization of the Golgi apparatus and the transport of proteins from one cisterna to the next. It is likely that the transport through the Golgi apparatus in the forward direction (red arrows) involves elements of (more...)
According to an alternative hypothesis, called the cisternal maturation model, the Golgi is viewed as a dynamic structure in which the cisternae themselves move through the Golgi stack. The vesicular tubular clusters that arrive from the ER fuse with one another to become a cis Golgi network, and this network then progressively matures to become a cis cisterna, then a medial cisterna, and so on. Thus, at the cis face of a Golgi stack, new cis cisternae would continually form and then migrate through the stack as they mature (Figure 13-30B). This model is supported by microscopic observations demonstrating that large structures such as collagen rods in fibroblasts and scales in certain algae—which are much too large to fit into classical transport vesicles—move progressively through the Golgi stack.
In the maturation model, the characteristic distribution of Golgi enzymes is explained by retrograde flow. Everything moves continuously forward with the maturing cisterna, including the processing enzymes that belong in the early Golgi apparatus. But budding COPI-coated vesicles continually collect the appropriate enzymes, almost all of which are membrane proteins, and carry them back to the earlier cisterna where they function. A newly formed cis cisterna would therefore receive its normal complement of resident enzymes primarily from the cisterna just ahead of it and would later pass them back to the next cis cisterna that forms.
As we discuss later, when a cisterna finally moves up to become part of the trans Golgi network, various types of coated vesicles bud off of it until this network disappears, to be replaced by a maturing cisterna just behind. At the same time, other transport vesicles are continually retrieving membrane from post-Golgi compartments and returning this membrane to the trans Golgi network.
The vesicular transport and the cisternal maturation model are not mutually exclusive. Indeed, evidence suggests that transport may occur by a combination of the two mechanisms, in which some cargo is moved forward rapidly in transport vesicles, whereas other cargo is moved forward more slowly as the Golgi apparatus constantly renews itself through cisternal maturation.
The unique architecture of the Golgi apparatus depends on both the microtubule cytoskeleton, as already discussed, and cytoplasmic Golgi matrix proteins, which form a scaffold between adjacent cisternae and give the Golgi stack its structural integrity. Some of the matrix proteins form long, filamentous tethers that are thought to help retain Golgi transport vesicles close to the organelle. When the cell prepares to divide, mitotic protein kinases phosphorylate the Golgi matrix proteins, causing the Golgi apparatus to fragment and disperse throughout the cytosol. During disassembly, Golgi enzymes are returned in vesicles to the ER, while other Golgi fragments are distributed to the two daughter cells. There, the matrix proteins are dephosphorylated, leading to the reassembly of the Golgi apparatus.
Remarkably, the Golgi matrix proteins can assemble into appropriately localized stacks near the centrosome even when Golgi membrane proteins are experimentally prevented from leaving the ER. This observation suggests that the matrix proteins are largely responsible for both the structure and location of the Golgi apparatus.
Correctly folded and assembled proteins in the ER are packaged into COPII-coated transport vesicles that pinch off from the ER membrane. Shortly thereafter the coat is shed and the vesicles fuse with one another to form vesicular tubular clusters, which move on microtubule tracks to the Golgi apparatus. Many resident ER proteins slowly escape, but they are returned to the ER from the vesicular tubular clusters and the Golgi apparatus by retrograde transport in COPI-coated vesicles.
The Golgi apparatus, unlike the ER, contains many sugar nucleotides, which are used by a variety of glycosyl transferase enzymes to perform glycosylation reactions on lipid and protein molecules as they pass through the Golgi apparatus. The N-linked oligosaccharides that are added to proteins in the ER are often initially trimmed by the removal of mannoses, and further sugars are added. Moreover, the Golgi is the site where O-linked glycosylation occurs and where glycosaminoglycan chains are added to core proteins to form proteoglycans. Sulfation of the sugars in proteoglycans and of selected tyrosines on proteins also occurs in a late Golgi compartment.
The Golgi apparatus distributes the many proteins and lipids that it receives from the ER and then modifies the plasma membrane, lysosomes, and secretory vesicles. It is a polarized structure consisting of one or more stacks of disc-shaped cisternae, each stack organized as a series of at least three functionally distinct compartments, termed cis, medial, and trans cisternae. The cis and trans cisternae are both connected to special sorting stations, called the cis Golgi network and the trans Golgi network, respectively. Proteins and lipids move through the Golgi stack in the cis-to-trans direction. This movement may occur by vesicular transport, by progressive maturation of the cis cisternae that migrate continuously through the stack, or by a combination of these two mechanisms. The enzymes that function in each particular region of the stack are thought to be kept there by continual retrograde vesicular transport from more distal cisternae. The finished new proteins end up in the trans Golgi network, which packages them in transport vesicles and dispatches them to their specific destinations in the cell.
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