Ch8. Cell Cytoplasmic Membrane Systems PDF

CHAPTER 8 Cytoplasmic Membrane Systems: Structure, Function, and Membrane Trafficking Source: C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus/Center for Disease Control and Prevention. Human immunodeficiency virus (HIV) on a human lymphocyte. This pseudocolored scanning electron microscopy image shows HIV particles (green) on the surface of a lymphocyte (pink). Many viruses, including HIV, must hijack the cells’ membrane systems in order to enter, propagate, and exit their host cells. CHAPTER OUTLINE 8.1 An Overview of the Endomembrane System 8.2 A Few Approaches to the Study of Endomembranes 8.3 The Endoplasmic Reticulum 8.4 The Golgi Complex 8.5 Types of Vesicle Transport The Human Perspective: Disorders Resulting from Defects in Lysosomal Function 8.6 Engineering Linkage: Extracellular Vesicles for Drug Delivery 8.7 Lysosomes 8.8 Green Cells: Plant Cell Vacuoles 8.9 The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior Experimental Pathways: Receptor-Mediated Endocytosis 8.10 Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts Hijacking the Cell Gaining access to an animal cell poses a formidable challenge to would-be invaders. Viruses, however, have evolved a multitude of ways to overcome cellular obstacles in order to execute their viral replication programs. Fundamental to the life cycle of all animal viruses is the hijacking of cellular membrane systems, including the plasma membrane, endosomal vesicles, endoplasmic reticulum (ER), and the Golgi complex. The first challenge that a virus faces is how to enter the cell. Some enveloped viruses, such as the herpes virus, fuse directly with the plasma membrane, depositing the genome-laden capsid inside the cell. A larger proportion of viruses, however, hijack endocytic pathways to enter the cell, thereby leaving few traces on the cell surface to alert the attention of roving immune cells. In these cases, viruses are internalized into vesicles known as endosomes and later release their genome into the cell. Another major challenge the virus now faces is how to replicate and produce new viable progeny. In recent years, researchers have found that cytoplasmic viruses often carry out an elaborate restructuring of host cell membranes, most often the ER membrane, to create new organelle-like structures, referred to as virus factories or viroplasm. Inside these virus factories, viral proteins required for replication are organized into assembly lines for the efficient production of new viral particles. In many ways, these factories operate like a scaled- down version of the cellular organelles from which they are derived, but are designed for the mass production of just a handful of molecules, rather than the thousands that the ER and Golgi complex are responsible for producing in any given cell. 8.1 An Overview of the Endomembrane System Under the light microscope, the cytoplasm of living cells appears relatively devoid of structure. Yet, even before the beginning of the twentieth century, examination of stained sections of animal tissues hinted at the existence of an extensive membrane network within the cytoplasm. It was not until the development of the electron microscope in the 1940s, however, that biologists began to appreciate the diverse array of membrane-bound structures present in the cytoplasm of most eukaryotic cells. These early electron microscopists saw membrane-bound vesicles of varying diameter containing material of different electron density, long channels bounded by membranes that radiate through the cytoplasm to form an interconnected network of canals, and stacks of flattened membrane-bound sacs. It became evident from these early electron microscopic studies, and the biochemical investigations that followed, that the cytoplasm of eukaryotic cells was subdivided into a variety of distinct compartments bounded by membrane barriers. As more types of cells were examined, it became apparent that these membranous compartments formed different organelles that could be identified in diverse cells from yeast to multicellular plants and animals. The extent to which the cytoplasm of a eukaryotic cell is occupied by membranous structures is illustrated in the electron micrograph of a maize root cell shown in Figure 8.1. As you will see in the following pages, each of these organelles contains a particular complement of proteins and is specialized for particular types of activities. Thus, just as a house or restaurant is divided into specialized rooms where different activities can take place independent of one another, the cytoplasm of a cell is divided into specialized membranous compartments. Keep in mind, as you examine the micrographs in this chapter, that these cytoplasmic organelles may appear as stable structures, like those rooms, but in fact they are dynamic compartments in continual flux. Source: Courtesy of Hilton H. Mollenhauer. FIGURE 8.1 Membrane-bound compartments of the cytoplasm. The cytoplasm of this root cap cell of a maize plant contains an array of membrane-bound organelles with structures and functions that will be examined in this chapter. As is evident in this micrograph, the combined surface area of the cytoplasmic membranes is many times greater than that of the surrounding plasma membrane. This chapter examines the structure and functions of the endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and vacuoles. Taken together, these organelles form an endomembrane system in which the individual components function as part of a coordinated unit. Mitochondria and chloroplasts are not part of this interconnected system and were the subjects of Chapters 5 and 6. Current evidence suggests that peroxisomes, which were also discussed in Chapter 6, have a dual origin; the basic elements of the boundary membrane are thought to arise from the endoplasmic reticulum, but many of the membrane proteins and the soluble internal proteins are taken up from the cytosol, as described in Section 8.10. The organelles of the endomembrane system are part of a dynamic, integrated network in which materials are shuttled back and forth from one part of the cell to another. For the most part, materials are shuttled between organelles—from the Golgi complex to the plasma membrane, for example—in small, membrane-bounded transport vesicles that bud from a donor membrane compartment (Figure 8.2a).1 Transport vesicles move through the cytoplasm in a directed manner, often pulled by motor proteins that operate on tracks formed by microtubules and microfilaments of the cytoskeleton (see Figure 9.1a). When it reaches its destination, a vesicle fuses with the membrane of the acceptor compartment, which receives the vesicle’s soluble cargo as well as its membranous wrapper (Figure 8.2a). Repeated cycles of budding and fusion shuttle a diverse array of materials along numerous pathways that traverse the cell. Vesicles are not the only way to transfer materials between endomembrane organelles, however. In recent years, it has become increasingly clear that the cell can build temporary tethers that connect two organelles in a dynamic and regulated way. These membrane contact sites have been found to be important regions for the transfer of different molecules, signaling, and communication. The best characterized are ER- mitchondria and ER-Golgi contact sites, which will be discussed later in the chapter. FIGURE 8.2 An overview of the biosynthetic/secretory and endocytic pathways that unite endomembranes into a dynamic, interconnected network. (a) Schematic diagram illustrating the process of vesicle transport by which materials are transported from a donor compartment to a recipient compartment. Vesicles form by membrane budding, during which specific membrane proteins (green spheres) of the donor membrane are incorporated into the vesicle membrane and specific soluble proteins (purple spheres) in the donor compartment are bound to specific receptors. When the transport vesicle subsequently fuses with another membrane, the proteins of the vesicle membrane become part of the recipient membrane, and the soluble proteins become sequestered within the lumen of the recipient compartment. (b) Materials follow the biosynthetic (or secretory) pathway from the endoplasmic reticulum, through the Golgi complex, and out to various locations including lysosomes, endosomes, secretory vesicles, secretory granules, vacuoles, and the plasma membrane. Materials follow the endocytic pathway from the cell surface to the interior by way of endosomes and lysosomes, where they are generally degraded by lysosomal enzymes. Several distinct pathways through the cytoplasm have been identified and are illustrated in the overview shown in Figure 8.2b. A biosynthetic pathway can be discerned in which proteins are synthesized in the endoplasmic reticulum, modified during passage through the Golgi complex, and transported from the Golgi complex to various destinations, such as the plasma membrane, a lysosome, or the large vacuole of a plant cell. This route is also referred to as the secretory pathway, because many of the proteins synthesized in the endoplasmic reticulum (as well as complex polysaccharides synthesized in the Golgi complex; Figure 7.35c) are destined to be discharged (secreted or exocytosed) from the cell. Secretory activities of cells can be divided into two types: constitutive and regulated (Figure 8.2b). During constitutive secretion, materials are transported in secretory vesicles from their sites of synthesis and discharged into the extracellular space in a continual manner. Most cells engage in constitutive secretion, a process that contributes not only to the formation of the extracellular matrix (Section 7.1), but also to the formation of the plasma membrane itself. During regulated secretion, materials are stored as membrane-bound packages and discharged only in response to an appropriate stimulus. Regulated secretion occurs, for example, in endocrine cells that release hormones, in pancreatic acinar cells that release digestive enzymes, and in nerve cells that release neurotransmitters. In some of these cells, materials to be secreted are stored in large, densely packed, membrane-bound secretory granules (see Figure 8.3). Proteins, lipids, and complex polysaccharides are transported through the cell along the biosynthetic or secretory pathway. The first part of this chapter focuses on the synthesis and transport of proteins, as summarized in Figure 8.2b. During the discussion, we consider several distinct classes of proteins. These include soluble proteins that are discharged from the cell, integral proteins of the various membranes depicted in Figure 8.2b, and soluble proteins that reside within the various compartments enclosed by the endomembranes (e.g., lysosomal enzymes). Whereas materials move out of the cell by the secretory pathway, the endocytic pathway operates in the opposite direction. By following the endocytic pathway, materials move from the outer surface of the cell to compartments, such as endosomes and lysosomes, located within the cytoplasm (Figure 8.2b). Source: (a) Courtesy of James D. Jamieson and George Palade. FIGURE 8.3 Autoradiography reveals the sites of synthesis and subsequent transport of secretory proteins. (a) Electron micrograph of a section of a pancreatic acinar cell that had been incubated for 3 minutes in radioactive amino acids and then immediately fixed and prepared for autoradiography. The black silver grains that appear in the emulsion following development are localized over the endoplasmic reticulum. (b–d) Diagrams of a sequence of autoradiographs showing the movement of labeled secretory proteins (represented by the silver grains in red) through a pancreatic acinar cell. When the cell is pulse-labeled for 3 minutes and immediately fixed (as shown in a), radioactivity is localized in the endoplasmic reticulum (b). After a 3-minute pulse and 17-minute chase, radioactive label is concentrated in the Golgi complex and adjacent vesicles (c). After a 3-minute pulse and 117-minute chase, radioactivity is concentrated in the secretory granules and is beginning to be released into the pancreatic ducts (d). The movement of vesicles and their contents along the various pathways of a cell is analogous to the movement of trucks carrying different types of cargo along the various highways of a city. Both types of transport require defined traffic patterns to ensure that materials are accurately delivered to the appropriate sites. For example, protein trafficking within a salivary gland cell requires the proteins of salivary mucus, which are synthesized in the endoplasmic reticulum, to be specifically targeted to secretory granules, while lysosomal enzymes, which are also manufactured in the endoplasmic reticulum, are specifically targeted to a lysosome. Different organelles also contain different integral membrane proteins. Consequently, membrane proteins must also be targeted to particular organelles, such as a lysosome or Golgi cisterna. These various types of cargo—secreted proteins, lysosomal enzymes, and membrane proteins—are routed to their appropriate cellular destinations by virtue of specific “addresses” or sorting signals that are encoded in the amino acid sequence of the proteins or in the attached oligosaccharides. The sorting signals are recognized by specific receptors that reside in the membranes or surface coats of budding vesicles, ensuring that the protein is transported to the appropriate destination. For the most part, the machinery responsible for driving this complex distribution system consists of soluble proteins that are recruited to specific membrane surfaces. During the course of this chapter, we will explain why one protein is recruited, for example, to the endoplasmic reticulum, whereas another protein might be recruited to a particular region of the Golgi complex. Great advances have been made over the past three decades in mapping the traffic patterns that exist in eukaryotic cells, identifying the specific addresses and receptors that govern the flow of traffic, and dissecting the machinery that ensures delivery to appropriate sites in the cell. Motor proteins and cytoskeletal elements, which play key roles in the movements of transport vesicles and other endomembranes, are described in the following chapter. We begin the study of endomembranes by discussing a few of the most important experimental approaches that have led to our current understanding. Review 1. Compare and contrast the biosynthetic pathway with the endocytic pathway. 2. How are particular proteins targeted to particular subcellular compartments? 8.2 A Few Approaches to the Study of Endomembranes Early studies with the electron microscope provided biologists with a detailed portrait of the structure of cells but gave them little insight into the functions of the components they were observing. Determining the functions of cytoplasmic organelles required the development of new techniques and the execution of innovative experiments. The experimental approaches described in the following sections have proven particularly useful in providing the foundation of knowledge on which current research on cytoplasmic organelles is based. Insights Gained from Autoradiography Among the many cells in the body, the acinar cells of the pancreas have a particularly extensive endomembrane system. These cells function primarily in the synthesis and secretion of digestive enzymes. After secretion from the pancreas, these enzymes are shipped through ducts to the small intestine, where they degrade ingested food matter. Where within the pancreatic acinar cells are the secretory proteins synthesized, and how do they reach the surface of the cells where they are discharged? These questions are inherently difficult to answer because all of the steps in the process of secretion occur simultaneously within the cell. To follow the steps of a single cycle from start to finish, that is, from the synthesis of a secretory protein to its discharge from the cell, James Jamieson and George Palade of Rockefeller University utilized the technique of autoradiography (Section 18.4). Autoradiography provides a means to visualize biochemical processes by allowing an investigator to determine the location of radioactively labeled materials within a cell. In this technique, tissue sections containing radioactive isotopes are covered with a thin layer of photographic emulsion, which is exposed by radiation emanating from radioisotopes within the tissue. Sites in the cells containing radioactivity are revealed under the microscope by silver grains in the overlying emulsion (Figure 8.3). To determine the sites where secretory proteins are synthesized, Palade and Jamieson incubated slices of pancreatic tissue in a solution containing radioactive amino acids for a brief period of time. During this period, labeled amino acids were taken up by the living cells and incorporated into the digestive enzymes as they were being synthesized on ribosomes. The tissues were quickly fixed, and the locations of proteins that had been synthesized with labeled amino acids during the brief incubation were determined autoradiographically. Using this approach, the endoplasmic reticulum was discovered to be the site of synthesis of secretory proteins (Figure 8.3a). To determine the intracellular path followed by secretory proteins from their site of synthesis to their site of discharge, Palade and Jamieson carried out an additional experiment. After incubating the tissue for a brief period in radioactive amino acids, they washed the tissue until it was free of excess isotope and transferred it to a medium containing only unlabeled amino acids. An experiment of this type is called a pulse-chase. The pulse refers to the brief incubation with radioactivity during which labeled amino acids are incorporated into protein. The chase refers to the period when the tissue is exposed to the unlabeled medium, a period during which additional proteins are synthesized using nonradioactive amino acids. The longer the chase, the farther the radioactive proteins manufactured during the pulse will have traveled from their site of synthesis within the cell. Using this approach, the movements of newly synthesized molecules can ideally be followed by observing a wave of radioactive material moving through the cytoplasmic organelles of cells from one location to the next until the process is complete. The results of these experiments—which first defined the biosynthetic (or secretory) pathway and tied a number of seemingly separate membranous compartments into an integrated functional unit—are summarized in Figure 8.3b–d. Insights Gained from the Use of Fluorescent Proteins The autoradiographic experiments described in the previous section require investigators to use an electron microscope to examine thin sections of different cells that have been fixed at various times after introduction of a radioactive label. Techniques involving the use of radioactive isotopes have largely been abandoned by modern cell biologists in favor of “tagging” proteins of interest using fluorescent proteins such as green fluorescent protein (GFP), which allows for viewing of protein movement in live cells under a light microscope (Section 18.1). Figure 8.4a,b shows a pair of micrographs depicting cells that contain a GFP-tagged protein. In this case, the cells were infected with a strain of the vesicular stomatitis virus (VSV) in which one of the viral genes (VSVG) is fused to the GFP gene. Viruses are useful in these types of studies because they turn infected cells into factories for the production of viral proteins, which are carried like any other protein cargo through the biosynthetic pathway. When a cell is infected with VSV, massive amounts of the VSVG protein are produced in the endoplasmic reticulum (ER). The VSVG molecules then traffic through the Golgi complex and are transported to the plasma membrane of the infected cell, where they are incorporated into viral envelopes. As in a radioactive pulse-chase experiment, the use of a virus allows investigators to follow a relatively synchronous wave of protein movement, in this case represented by a wave of green fluorescence that begins soon after infection. Synchrony can be enhanced, as was done in the experiment depicted in Figure 8.4, by using a virus with a mutant VSVG protein that is unable to leave the ER of infected cells grown at an elevated temperature (e.g., 40°C). When the temperature is lowered to 32°C, the fluorescent GFP- VSVG protein that had accumulated in the ER (Figure 8.4a,c) moves synchronously to the Golgi complex (Figure 8.4b,c), where various processing events occur, and then on to the plasma membrane. Mutants of this type that function normally at a reduced (permissive) temperature, but not at an elevated (restrictive) temperature, are described as temperature- sensitive mutants. An experiment utilizing two different fluorescent probes is described in the Experimental Pathways feature in Section 8.9. Source: (a, b) ©1999 Daniel S. et al. Originally published in The Journal of Cell Biology. http://doi.org/10.1083/jcb.144.5.869 FIGURE 8.4 The use of green fluorescent protein (GFP) reveals the movement of proteins within a living cell. (a) Fluorescence micrograph of a live cultured mammalian cell that had been infected with the VSV at 40°C. This particular strain of the VSV contained a VSVG gene that (1) was fused to a gene encoding the GFP and (2) contained a temperature-sensitive mutation that prevented the newly synthesized VSVG protein from leaving the ER when kept at 40°C. The green fluorescence in this micrograph is restricted to the ER. (b) Fluorescence micrograph of a live infected cell that was held at 40°C to allow the VSVG protein to accumulate in the ER and then incubated at 32°C for 10 minutes. The fluorescent VSVG protein has moved on to the Golgi complex. (c) Schematic drawing showing the retention of the mutant VSVG protein in the ER at 40°C and its synchronous movement to the Golgi complex within 10 minutes of incubation at the lower temperature. In recent years, new technological developments, including multispectral imaging (visualizing multiple different-colored fluorophores simultaneously), super resolution microscopy, and lattice light sheet microscopy (see Chapter 18), have enabled researchers to better understand the complex dynamics and interrelationships of endomembrane systems in live cells. For example, these imaging technologies have been used to visualize the dynamic interactions of different organelles in a cell (Figure 8.5). Source: Reprinted by permission from Springer Nature: Alex M. Valm et al. Nature 546, pages 162–167, 2017. FIGURE 8.5 Microscopic images of a mammalian cell expressing multiple fluorophores to visualize different organelles. Peroxisomes (perox), mitochondria (mito), ER, Golgi, lysosomes (lyso) and lipid droplets (LD) were visualized simultaneously in a living cell and analyzed. The right image a composite of all the fluorophores (CFP, EGFP and YEP), providing a snapshot of how different organelles are organized in the cell relative to one another. Insights Gained from the Analysis of Subcellular Fractions Electron microscopy, autoradiography, and the use of fluorescent tags provide information on the structure and function of cellular organelles but fail to provide much insight into the molecular composition of these structures. Techniques to break up (homogenize) cells and isolate particular types of organelles were pioneered in the 1950s and 1960s by Albert Claude and Christian De Duve. When a cell is ruptured by homogenization, the cytoplasmic membranes become fragmented and the fractured edges of the membrane fragments fuse to form spherical vesicles less than 100 nm in diameter. Vesicles derived from different organelles (nucleus, mitochondrion, plasma membrane, endoplasmic reticulum, and so forth) have different properties, which allow them to be separated from one another, an approach that is called subcellular fractionation. Membranous vesicles derived from the endomembrane system (primarily the ER and Golgi complex) form a heterogeneous collection of similar-sized vesicles referred to as microsomes. A rapid (and crude) preparation of the microsomal fraction of a cell is depicted in Figure 8.6a. The microsomal fraction can be further fractionated into smooth and rough membrane fractions (Figure 8.6b,c) by the gradient techniques discussed in Section 18.6. Once isolated, the biochemical composition of various fractions can be determined. In recent years, the identification of the proteins present in cell fractions has been carried out using proteomic technology. Once a particular organelle has been isolated, the proteins can be extracted, separated, and identified by mass spectrometry as discussed in Section 18.7. Hundreds of proteins can be identified simultaneously, providing a comprehensive molecular portrait of any organelle that can be prepared in a relatively pure state. In one example of this technology, it was found that a simple phagosome (Section 8.9) containing an ingested latex bead comprised more than 160 different proteins, many of which had never previously been identified or were not known to be involved in phagocytosis. Source: (b–c) Courtesy of J. A. Higgins and R. J. Barnett. FIGURE 8.6 Isolation of a microsomal fraction by differential centrifugation. (a) When a cell is broken by mechanical homogenization (step 1), the various membranous organelles become fragmented and form spherical membranous vesicles. Vesicles derived from different organelles can be separated by various techniques of centrifugation. In the procedure depicted here, the cell homogenate is first subjected to low-speed centrifugation to pellet the larger particles, such as nuclei and mitochondria, leaving the smaller vesicles (microsomes) in the supernatant (step 2). The microsomes can be removed from the supernatant by centrifugation at higher speeds for longer periods of time (step 3). A crude microsomal fraction of this type can be fractionated into different vesicle types in subsequent steps. (b) Electron micrograph of a smooth microsomal fraction in which the membranous vesicles lack ribosomes. (c) Electron micrograph of a rough microsomal fraction containing ribosome-studded membranes. Insights Gained from the Use of Cell-Free Systems Once techniques to fractionate membranous organelles were developed, researchers began to probe the capabilities of these crude subcellular preparations. They found that the isolated parts of a cell were capable of remarkable activities. These early cell-free systems—so- called because they did not contain whole cells—provided a wealth of information about biological processes that were impossible to study within the complex environment of intact cells. During the 1960s, for example, George Palade, Philip Siekevitz, and their colleagues at Rockefeller University set out to learn more about the properties of the rough microsomal fraction (shown in Figure 8.6c), with membrane vesicles derived from the rough ER (Section 8.3). They found that they could strip a rough microsomal preparation of its attached particles, and the isolated particles (i.e., ribosomes) were capable of synthesizing proteins when provided with required ingredients from the cytosol. Under these conditions, the newly synthesized proteins were simply released by the ribosomes into the aqueous fluid of the test tube. When the same experiment was carried out using intact rough microsomes, the newly synthesized proteins were no longer released into the incubation medium but were trapped within the lumen of the membranous vesicles. It was concluded from these studies that the microsomal membrane was not required for the incorporation of amino acids into proteins but for sequestering newly synthesized secretory proteins within the ER cisternal space. Over the past few decades, researchers have used cell-free systems to identify the roles of many of the proteins involved in membrane trafficking. James Rothman and Randy Schekman, recipients of the 2013 Nobel Prize in Physiology or Medicine, have been noted for their use of cell-free systems in their research on vesicle trafficking. Figure 8.7 shows a liposome with vesicles budding from its surface (arrows) from an experiment conducted in Schekman’s laboratory. As discussed in Section 4.2, liposomes are vesicles with a surface consisting of an artificial bilayer created in the laboratory from purified phospholipids. The buds and vesicles seen in Figure 8.7 were produced after the preparation of liposomes was incubated with purified proteins. These proteins normally comprise coats on the cytosolic surface of transport vesicles within the cell. Without the added coat proteins, vesicle budding could not occur. Source: Courtesy of Lelio Orci and Randy Schekman. FIGURE 8.7 Formation of coated vesicles in a cell-free system. Electron micrograph of a liposome preparation that had been incubated with the components required to promote vesicle budding within the cell. The proteins in the medium have become attached to the surface of the liposomes and have induced the formation of protein-coated buds (arrows). Using this strategy, in which cellular processes are reconstituted in vitro from purified components, researchers have been able to study the proteins that bind to the membrane to initiate vesicle formation, those responsible for cargo selection, and those that sever the vesicles from the donor membrane. Insights Gained from the Study of Mutant Phenotypes A mutant is an organism (or cultured cell) with chromosomes containing one or more genes that encode abnormal proteins. When a protein encoded by a mutant gene is unable to carry out its normal function, the cell carrying the mutation exhibits a characteristic deficiency. Determining the precise nature of the deficiency provides information about the function of the normal protein. Large-scale studies of the genetic basis of secretion have been carried out largely on yeast cells, most notably by Randy Schekman and colleagues. In yeast, as in all eukaryotic cells, vesicles bud from the ER and travel to the Golgi complex, where they fuse with the Golgi cisternae (Figure 8.8a). To identify genes coding for proteins involved in this portion of the secretory pathway (i.e., sec genes), researchers screen for mutant cells that exhibit an abnormal distribution of cytoplasmic membranes. An electron micrograph of a wild-type yeast cell is shown in Figure 8.8b. The cell depicted in Figure 8.8c has a mutation in a gene that encodes a protein involved in the formation of vesicles at the ER membrane (step 1, Figure 8.8a). In the absence of vesicle formation, mutant cells accumulate an expanded ER. In contrast, the cell depicted in Figure 8.8d carries a mutation in a gene that encodes a protein involved in vesicle fusion (step 2, Figure 8.8a). When this gene product is defective, the mutant cells accumulate an excess number of unfused vesicles. Researchers have isolated dozens of different mutants, which, taken as a group, exhibit disruptions in virtually every step of the secretory pathway. The genes responsible for these defects have been cloned and sequenced, and the proteins they encode have been isolated. Isolation of proteins from yeast has launched successful searches for homologous proteins (i.e., proteins with related sequences) in mammals. Source: From Chris A. Kaiser and Randy Schekman, Cell 61:724, 1990; Elsevier. FIGURE 8.8 The use of genetic mutants in the study of secretion. (a) The first leg of the biosynthetic secretory pathway in budding yeast. The steps are described in the narrative. (b) Electron micrograph of a section through a wild-type yeast cell. (c) A yeast cell bearing a mutation in the sec12 gene with a product involved in the formation of vesicles at the ER membrane (step 1, part a). Because vesicles cannot form, expanded ER cisternae accumulate in the cell. (d) A yeast cell bearing a mutation in the sec17 gene, with a product involved in vesicle fusion (step 2, part a). Because they cannot fuse with Golgi membranes, the vesicles (indicated by the arrowheads) accumulate in the cell. [The mutants depicted in c and d are temperature-sensitive mutants. When kept at the lower (permissive) temperature, they are capable of normal growth and division.] One of the most important lessons learned from the use of all of these techniques is that the dynamic activities of the endomembrane system are highly conserved. Not only do yeast, plant, insect, and human cells carry out similar processes, but they do so with remarkably similar proteins. It is evident that the structural diversity of cells belies their underlying molecular similarities. In many cases, proteins from widely divergent species are interchangeable. For example, cell-free systems derived from mammalian cells can often utilize yeast proteins to facilitate vesicle transport. Conversely, yeast cells with genetic deficiencies that disrupt some phase of the biosynthetic pathway can often be “cured” by genetically engineering them to carry mammalian genes. Over the past two decades, researchers interested in searching for genes that affect a particular cellular process in plant or animal cells have taken advantage of a cellular phenomenon called RNA interference (RNAi). RNAi is a process in which cells produce small RNAs (called siRNAs) that bind to specific mRNAs and inhibit the translation of these mRNAs into proteins. This phenomenon and its use are discussed in detail in Sections 11.5 and 18.17. For the present purpose, simply note that researchers can synthesize a collection (library) of siRNAs capable of inhibiting the translation of virtually any mRNA that is produced by a genome. Each mRNA represents the expression of a specific gene; therefore, the genes involved in a particular process can be found by determining which siRNAs interfere with that process. In the experiment depicted in Figure 8.9, researchers set out to identify genes involved in various steps of the secretory pathway, a goal similar to that of the investigators studying the yeast mutants shown in Figure 8.8. In this case, researchers used a strain of cultured Drosophila cells and attempted to identify genes that affected the localization of mannosidase II, an enzyme synthesized in the ER that moves via transport vesicles to the Golgi complex, where it takes up residence. Figure 8.9a shows a control cell that is synthesizing a GFP-labeled version of mannosidase II; as expected, the fluorescence becomes localized in the numerous Golgi complexes of the cell. Figure 8.9b shows a cell containing siRNA molecules that have caused a relocation of the GFP-mannosidase into the ER, which is seen to be fused with the Golgi complexes. This phenotype is typically caused by the absence of one of the proteins involved in the transport of the enzyme from the ER to the Golgi complex. Of the 130 different siRNAs that were found to interfere in some way with the secretory pathway in this study, 31 of them generated a phenotype similar to that shown in Figure 8.9b. Included among these 31 siRNAs were numerous species that inhibited the expression of genes already known to be involved in the secretory pathway. In addition, the study identified other genes with unknown function that are now presumed to be involved in these processes as well. In recent years, CRISPR-based technologies have been developed that also allow for silencing and editing of genes in a specific and high-throughput manner. These methods, which have begun to eclipse RNAi-based methods for gene silencing, are discussed further in Section 18.17. Source: (a, b) Reprinted by permission from Springer Nature: F. Bard et al. Nature 439, pages 604–607, 2006. FIGURE 8.9 Inhibition of gene expression with RNA interference. (a) A control Drosophila S2 cultured cell expressing GFP-labeled mannosidase II. The fluorescent enzyme becomes localized in the Golgi complex after its synthesis in the ER. (b) A cell that has been genetically engineered to express a specific siRNA, which binds to a complementary mRNA and inhibits translation of the encoded protein. In this case, the siRNA has caused the fluorescent enzyme to remain in the ER, which has fused with the Golgi membranes. This phenotype suggests that the mRNA being affected by the siRNA encodes a protein involved in an early step of the secretory pathway during which the enzyme is synthesized in the ER and traffics to the Golgi complex. Among the genes that exhibit this phenotype when targeted by siRNAs are those encoding proteins of the COPI coat, Sar1, and Sec23. The functions of these proteins are discussed later in the chapter. Review 1. Describe the differences between an autoradiograph of a pancreas cell that had been incubated in labeled amino acids for 3 minutes and immediately fixed and one of a cell that had been labeled for 3 minutes, chased for 40 minutes, and then fixed. 2. What techniques or approaches might you use to learn which proteins are normally present in the endoplasmic reticulum? 3. How does the isolation of a mutant yeast that accumulates vesicles provide information on the process of protein trafficking? 4. How can GFP be used to study membrane dynamics? 8.3 The Endoplasmic Reticulum The endoplasmic reticulum (ER) comprises a network of membranes that penetrate much of the cytoplasm. The ER probably evolved from invaginations of the plasma membrane as described in Section 1.4. Enclosed within the ER is an extensive space, or lumen, that is separated from the surrounding cytosol by the ER membrane. As will be evident in the following discussion, the composition of the luminal (or cisternal) space inside the ER membranes is quite different from that of the surrounding cytosolic space. Like other subcellular organelles, the ER is a highly dynamic structure undergoing continual turnover and reorganization. The ER is divided into two subcompartments: the rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum (SER). The RER is defined by the presence of ribosomes bound to its cytosolic surface, whereas the SER lacks associated ribosomes. The RER is typically composed of a network of flattened sacs (cisternae), as shown in Figure 8.9a and 8.9c, where each layer is connected to its neighbors by helicoidal membranes (Figure 8.10b). The RER is continuous with the outer membrane of the nuclear envelope, which also bears ribosomes on its cytosolic surface (Figure 8.2b). In contrast, the membranes of the SER are highly curved and tubular, forming an interconnecting system of pipelines traversing the cytoplasm (Figure 8.10a). When cells are homogenized, the SER tubules fragment into smooth-surfaced vesicles, whereas the RER sheets fragment into rough- surfaced vesicles (Figure 8.6b,c). Source: (b) From K. Tanaka, Int. Rev. Cytol. 68:1010, 1980. Courtesy K. Tanaka; (c) From M. Terasaki et al., Cell 154, issue 2 p285–296, 2013, with permission from Elsevier; (d) From George H. Patterson, Cell Image Library, CIL722. FIGURE 8.10 The rough endoplasmic reticulum (RER). (a) Schematic diagram showing the stacks of flattened cisternae that make up the RER. The cytosolic surface of the ER membrane contains bound ribosomes, which give the cisternae their rough appearance. (b) Scanning electron micrograph of the RER in a pancreatic acinar cell. (c) High-resolution EM imaging has revealed that ER sheets are connected by helicoidal ramps. A three-dimensional reconstruction of such a ramp is shown here. (d) Visualization of the ER in a whole cultured live mammalian cell as revealed by GFP fluorescence of an ER protein. Fluorescently labeled proteins and lipids are capable of diffusing from one type of ER into the other, indicating that their membranes are continuous. In fact, the two types of ER share many of the same proteins and engage in certain common activities, such as the synthesis of certain lipids and cholesterol. At the same time, numerous proteins are found only in one or the other type of ER. For example, the high degree of curvature of the SER tubules is induced and maintained by the presence of large numbers of membrane-bending proteins, called reticulons, which are only present in small numbers at the edges of the flattened RER sheets. The RER, in contrast, contains high levels of proteins involved in the movement of nascent proteins into the ER lumen. Different types of cells contain markedly different ratios of the two types of ER, depending on the activities of the cell. For example, cells that secrete large amounts of proteins, such as the cells of the pancreas or salivary glands, have extensive regions of RER (Figure 8.10b–d). We will return to the function of the RER shortly, but first we describe the activities of the SER. The Smooth Endoplasmic Reticulum The SER is extensively developed in a number of cell types, including those of skeletal muscle, kidney tubules, and steroid-producing endocrine glands (Figure 8.11). SER functions include Source: DON W. FAWCETT/Science Source FIGURE 8.11 The smooth ER (SER). Electron micrograph of a Leydig cell from the testis showing the extensive SER where steroid hormones are synthesized. Synthesis of steroid hormones in the endocrine cells of the gonad and adrenal cortex. Detoxification in the liver of a wide variety of organic compounds, including barbiturates and ethanol, chronic use of which can lead to proliferation of the SER in liver cells. Detoxification is carried out by a collection of oxygen-transferring enzymes (oxygenases), including the cytochrome P450 family. These enzymes are noteworthy for their lack of substrate specificity; they are able to oxidize thousands of different hydrophobic compounds and convert them into more hydrophilic, more readily excreted derivatives. The results are not always positive. For example, the relatively harmless compound benzo[a]pyrene formed when meat is charred on a grill is converted into a potent carcinogen by the “detoxifying” enzymes of the SER. Cytochrome P450s metabolize many prescribed medications, and genetic variation in these enzymes among humans may explain differences from one person to the next in the effectiveness and side effects of many drugs. Sequestering calcium ions within the cytoplasm of cells. The regulated release of Ca2+ from the SER of skeletal and cardiac muscle cells (known as the sarcoplasmic reticulum in muscle cells) triggers contraction. The Rough Endoplasmic Reticulum Early investigations into the functions of the RER were carried out on cells that secrete large quantities of proteins, such as the acinar cells of the pancreas (Figure 8.3) or the mucus- secreting cells of the lining of the digestive tract (Figure 8.12). It is evident from the drawing (Figure 8.12a) and micrograph (Figure 8.12b) that the organelles of these epithelial secretory cells are positioned in the cell in such a way as to produce a distinct polarity from one end to the other. The nucleus and an extensive array of RER cisternae are located near the basal surface of the cell, which faces the blood supply. The Golgi complex is located in the central region of the cell. The apical surface of the cell faces a duct that carries the secreted proteins out of the organ. The cytoplasm at the apical end of the cell is filled with secretory granules with contents ready to be released into the duct upon arrival of the appropriate signal. The polarity of these glandular epithelial cells reflects the movement of secretory proteins through the cell from their site of synthesis to their site of discharge. The RER is the starting point of the biosynthetic pathway: It is the site of synthesis of the proteins, carbohydrate chains, and phospholipids that journey through the membranous compartments of the cell. Source: (a) Adapted from Marian Neutra and C. P. Leblond, Copyright 1966, Rockefeller University Press. Originally published in The Journal of Cell Biology Volume 30:119. (b) From Alain Rambourg and Yves Clermont. American Journal of Anatomy 179, Issue 2, 1987. This material is used by permission of John Wiley & Sons Inc. FIGURE 8.12 Polarized structure of a secretory cell. (a) Drawing of a mucus- secreting goblet cell from the rat colon. (b) Low-power electron micrograph of a mucus-secreting cell from Brunner’s gland of the mouse small intestine. Both types of cells display a distinctly polarized arrangement of organelles, reflecting their role in secreting large quantities of mucoproteins. The basal ends of the cells contain the nucleus and RER. Proteins synthesized in the RER move into the closely associated Golgi complex and from there into membrane-bound carriers, in which the final secretory product is concentrated. The apical regions of the cells are filled with secretory granules containing the mucoproteins ready for release into a duct. The functions of the RER include the synthesis of proteins and the synthesis of most of the lipids of a cell’s membranes. The addition of sugars to the asparagine residues of proteins begins in the RER and continues in the Golgi complex. The following sections present the RER’s role in the synthesis of secreted proteins, lysosomal proteins, and integral membrane proteins. Synthesis of Proteins on Membrane-Bound versus Free Ribosomes The discovery of the RER as the site of synthesis of secretory proteins in pancreatic acinar cells was described earlier (Figure 8.3). Similar results were found for other types of secretory cells, including intestinal goblet cells that secrete mucoproteins, endocrine cells that secrete polypeptide hormones, plasma cells that secrete antibodies, and liver cells that secrete blood serum proteins. Further experiments revealed that polypeptides are synthesized at two distinct locales within the cell: 1. Approximately one-third of the proteins encoded by a mammalian genome are synthesized on ribosomes attached to the cytosolic surface of the RER membranes and released into the ER lumen in a process called co-translational translocation. These include (1) secreted proteins, (2) integral membrane proteins, and (3) soluble proteins that reside within compartments of the endomembrane system, including the ER, Golgi complex, lysosomes, endosomes, vesicles, and plant vacuoles. 2. Other polypeptides are synthesized on free ribosomes, ribosomes that are not attached to the RER, and are subsequently released into the cytosol. This class includes (1) proteins destined to remain in the cytosol (such as the enzymes of glycolysis and the proteins of the cytoskeleton), (2) peripheral proteins of the cytosolic surface of membranes (such as spectrins and ankyrins that are only weakly associated with the plasma membrane’s cytosolic surface), (3) proteins that are transported to the nucleus (Section 12.3), and (4) proteins to be incorporated into peroxisomes, chloroplasts, and mitochondria. Proteins in the latter two groups are synthesized to completion in the cytosol and then imported posttranslationally into the appropriate organelle across its boundary membrane(s) (Section 8.10). What determines the location in a cell where a protein is synthesized? In the early 1970s, Günter Blobel, in collaboration with David Sabatini and Bernhard Dobberstein of Rockefeller University, first proposed, and then demonstrated, that the site of synthesis of a protein is determined by the sequence of amino acids in the N-terminal portion of the polypeptide, which is the first part to emerge from the ribosome during protein synthesis. They suggested the following: 1. Secretory proteins contain a signal sequence at their N-terminus that directs the emerging polypeptide and ribosome to the ER membrane. 2. The polypeptide moves into the cisternal space of the ER through a protein-lined, aqueous channel in the ER membrane. It was proposed that the polypeptide moves through the membrane as it is being synthesized, that is, co-translationally.2 This proposal, known as the signal hypothesis, has been substantiated by a large body of experimental evidence. Even more important, Blobel’s original concept that proteins contain built-in “address codes” has been shown to apply in principle to virtually all types of protein trafficking pathways throughout the cell. Synthesis of Secretory, Lysosomal, or Plant Vacuolar Proteins The synthesis of secretory, lysosomal, or plant vacuolar proteins usually takes place by co- translational translocation. In this process, newly forming proteins are deposited into the ER lumen by a ribosome attached to the ER membrane (Figure 8.13). The synthesis of the polypeptide begins after a messenger RNA binds to a free ribosome. In fact, all ribosomes are thought to be identical; those employed in the synthesis of secretory, lysosomal, or plant vacuolar proteins are not distinguishable from those used for production of proteins that remain in the cytosol. Polypeptides that undergo co-translational translocation contain a signal sequence—which typically includes a stretch of 6–15 hydrophobic amino acid residues—that targets the nascent polypeptide to the ER membrane and leads to the compartmentalization of the polypeptide within the ER lumen. (A nascent polypeptide is one in the process of being synthesized.) Although the signal sequence is usually located at or near the N-terminus, it occupies an internal position in some polypeptides. Source: (b) Reprinted by permission from Macmillan Publishers Ltd: From Tom A. Rapoport, Nature 450:664, 2007; ©2007. (c) From Thomas Becker et al., Science, Vol 326, 1372, 2009, Figure 6e. Roland Beckmann, University of Munich. ©2009. Reprinted with permission from American Association for the Advancement of Science AAAS. FIGURE 8.13 A schematic model of the synthesis of a secretory protein (or a lysosomal enzyme) on a membrane-bound ribosome of the RER. (a) Synthesis of the polypeptide begins on a free ribosome. As the signal sequence (shown in red) emerges from the ribosome, it binds to the SRP (step 1), which stops further translation until the SRP–ribosome–nascent chain complex can make contact with the ER membrane. The SRP–ribosome complex then collides with and binds to an SRP receptor (SR) situated within the ER membrane (step 2). Attachment of this complex to the SRP receptor is followed by the release of the SRP and the association of the ribosome with a translocon of the ER membrane (step 3). These latter events are accompanied by the reciprocal hydrolyis of GTP molecules (not shown) bound to both the SRP and its receptor. In the model depicted here, the signal peptide then binds to the interior of the translocon, displacing the plug from the channel and allowing the remainder of the polypeptide to translocate through the membrane cotranslationally (step 4). After the nascent polypeptide passes into the lumen of the ER, the signal peptide is cleaved by a membrane protein (the signal peptidase, not shown), and the protein undergoes folding with the aid of ER chaperones, such as BiP. Studies suggest that translocons are organized into groups of two or four units rather than singly as shown here. (b) Cross- sectional view of the translocon channel from the side based on the X-ray crystal structure of an archaebacterial translocon. The hourglass shape of the aqueous channel and its helical plug are evident. The ring of hydrophobic side chains (green) situated at the narrowest site within the channel is also shown. (c) Representation of a ribosome–translocon complex in the act of synthesis and translocation of a nascent protein based on cryo-EM (Section 18.14). The exit channel within the ribosome is seen to be aligned with the conducting channel within the translocon. PCC, protein conducting channel; NC, nascent chain; P-tRNA, peptidyl-tRNA; 40S and 60S, ribosomal subunits. As it emerges from the ribosome, the hydrophobic signal sequence is recognized by a signal recognition particle (SRP), which consists in mammalian cells of six distinct polypeptides and an RNA molecule, called the 7SL RNA. The SRP binds to both the signal sequence on the nascent polypeptide and the ribosome (step 1, Figure 8.13a), temporarily arresting further synthesis of the polypeptide. The SRP–ribosome–nascent polypeptide complex is then recruited to the ER membrane through an interaction between the SRP and the SRP receptor on the ER membrane (step 2). The ribosome (with its nascent polypeptide) is then handed off from the SRP to the translocon (step 3). The translocon is a protein channel embedded in the ER membrane through which the nascent polypeptide is able to move in its passage from the ribosome to the ER lumen. X-ray crystallography of the translocon (Figure 8.13b) has provided insight into how translocation occurs. These studies have revealed the presence within the translocon of a pore in the shape of an hourglass with a ring of six hydrophobic amino acids situated at its narrowest diameter. In the inactive (i.e., non-translocating) state, the opening in the pore ring is plugged by a short α helix. This plug is thought to seal the channel, preventing the unwanted passage of calcium and other ions between the cytosol and the ER lumen. Upon attachment of the ribosome to the translocon, the signal sequence is recognized, and the nascent polypeptide is inserted into the narrow aqueous channel of the translocon (step 3). It is proposed that contact of the signal sequence with the interior of the translocon leads to displacement of the plug and opening of the passageway. The growing polypeptide is then translocated through the hydrophobic pore ring and into the ER lumen (step 4 and Figure 8.13c). Because the pore ring observed in the crystal structure has a diameter (5–8 Å) that is considerably smaller than that of a helical polypeptide chain, it is thought that the channel is able to open in a hinge-like manner as the nascent chain traverses the channel. Upon termination of translation and passage of the completed polypeptide through the translocon, the membrane-bound ribosome is released from the ER membrane, and the helical plug is reinserted into the translocon channel. Several of the steps involved in the synthesis and trafficking of secretory proteins are regulated by the binding or hydrolysis of GTP. As will be discussed at length in Chapter 15 and elsewhere in this chapter, GTP-binding proteins (or G proteins) play key regulatory roles in many different cellular processes. G proteins can be present in at least two alternate conformations, one containing a bound GTP molecule and the other a bound GDP molecule. GTP- and GDP-bound versions of a G protein have different conformations and thus have different abilities to bind other proteins. Because of this difference in binding properties, G proteins act like “molecular switches”; the GTP-bound protein typically turns the process on, and hydrolysis of the bound GTP turns it off. Among the components depicted in Figure 8.13, both the SRP and the SRP receptor are G proteins that interact with one another in their GTP-bound states. Prior to GTP hydrolysis, the G protein complex undergoes a dramatic conformational change that simultaneously exposes the signal sequence and makes room for the translocon to bind to the ribosome. The hydrolysis of GTP bound to these two proteins triggers the release of the signal sequence by the SRP. Processing of Newly Synthesized Proteins in the Endoplasmic Reticulum As it enters the RER cisterna, a nascent polypeptide is acted on by a variety of enzymes located within either the membrane or the lumen of the RER. The N-terminal portion containing the signal peptide is removed from most nascent polypeptides by a proteolytic enzyme, the signal peptidase. Carbohydrates are added to the nascent protein by the enzyme oligosaccharyltransferase (discussed later in this section). Both the signal peptidase and oligosaccharyltransferase are integral membrane proteins associated with the translocon that act on nascent proteins as they enter the ER lumen. The RER is a major protein processing plant. To meet its obligations, the RER lumen is packed with molecular chaperones that recognize and bind to unfolded or misfolded proteins and give them the opportunity to attain their correct (native) three-dimensional structure (described later in this section). The ER lumen also contains a number of protein-processing enzymes, such as protein disulfide isomerase (PDI). Proteins enter the ER lumen with their cysteine residues in the reduced (⏤SH) state, but they leave the compartment with many of these residues joined to one another as oxidized disulfides (⏤SS⏤) (Section 2.7). The formation (and rearrangement) of disulfide bonds is catalyzed by PDI. Disulfide bonds play an important role in maintaining the stability of proteins that are present at the extracellular surface of the plasma membrane or secreted into the extracellular space. The ER is ideally constructed for its role as a port of entry for the biosynthetic pathway of the cell. Its membrane provides a large surface area to which many ribosomes can attach (an estimated 13 million per liver cell). The lumen of the ER cisternae provides a specialized local environment that favors the modification, folding, and assembly of a selected subset of the cell’s proteins. The segregation of these newly synthesized proteins in the ER cisternae removes them from the cytosol and allows them to be modified and dispatched toward their ultimate destination, whether outside the cell or within one of the cytoplasm’s membranous organelles. Synthesis of Integral Membrane Proteins on ER-Bound Ribosomes Integral membrane proteins—other than those of mitochondria and chloroplasts—are also synthesized by co-translational translocation using the same machinery described for the synthesis of secretory and lysosomal proteins (Figure 8.13). However, unlike soluble secretory and lysosomal proteins, which pass entirely through the ER membrane during translocation, integral proteins contain one or more hydrophobic transmembrane segments (Section 4.3) that are shunted directly from the channel of the translocon into the lipid bilayer. How can such a transfer take place? Structural studies of the translocon already described showed the translocon to have a clam- shaped conformation with a groove or seam along one side of the wall where the channel might open and close. As a polypeptide passes through the translocon, it is proposed that this lateral “gate” in the channel continually opens and closes, which gives each segment of the nascent polypeptide an opportunity to partition itself according to its solubility properties into either the aqueous compartment within the translocon channel or the surrounding hydrophobic core of the lipid bilayer. Those segments of the nascent polypeptide that are sufficiently hydrophobic will spontaneously “dissolve” into the lipid bilayer and ultimately become transmembrane segments of an integral membrane protein. This concept has received strong support from an in vitro study in which translocons were given the opportunity to translocate custom-designed nascent proteins containing test segments of varying hydrophobicity. The more hydrophobic the test segment, the greater the likelihood it would pass through the wall of the translocon and become integrated as a transmembrane segment of the bilayer. Figure 8.14 shows the synthesis of a pair of integral membrane proteins containing a single transmembrane segment. Single-spanning membrane proteins can be oriented with their N-terminus facing either the cytosol or the lumen of the ER (and eventually the extracellular space). As noted in Section 4.3, the most common determinant of membrane protein alignment is the presence of positively charged amino acid residues flanking the cytosolic end of a transmembrane segment (see Figure 4.18). During the synthesis of membrane proteins, the inner lining of the translocon is thought to orient the nascent polypeptide, as indicated in Figure 8.14, so that the more positive end faces the cytosol. In multispanning proteins (as shown in Figure 4.32d), sequential transmembrane segments typically have opposite orientations. The arrangement of these proteins within the membrane is determined by the direction in which the first transmembrane segment is inserted. Once that has been determined, every other transmembrane segment must be rotated 180° before it can exit the translocon. Studies performed with purified components in cell-free systems suggest that a translocon, by itself, is capable of properly orienting transmembrane segments. It would appear that the translocon is more than a simple passageway through the ER membrane; it is a complex machine capable of recognizing various signal sequences and performing complex mechanical activities. FIGURE 8.14 A schematic model for the synthesis of an integral membrane protein that contains a single transmembrane segment near the N-terminus of the nascent polypeptide. The SRP and the various components of the membrane that were shown in Figure 8.13 are also involved in the synthesis of integral proteins, but they have been omitted for simplicity. The nascent polypeptide enters the translocon just as if it were a secretory protein (step 1). However, the entry of the hydrophobic transmembrane sequence into the pore blocks further translocation of the nascent polypeptide through the channel. Steps 2 and 3 show the synthesis of a transmembrane protein with an N-terminus in the lumen of the ER and a C-terminus in the cytosol. In step 2, the lateral gate of the translocon has opened and expelled the transmembrane segment into the bilayer. Step 3 shows the final disposition of the protein. Steps 2a–4a show the synthesis of a transmembrane protein with a C-terminus in the lumen and an N-terminus in the cystosol. In step 2a, the translocon has reoriented the transmembrane segment, in keeping with its reversed positively and negatively charged flanks. In step 3a, the translocon has opened laterally and expelled the transmembrane segment into the bilayer. Step 4a shows the final disposition of the protein. White-colored + and – signs indicate the proposed charge displayed by the inner lining of the translocon. The difference in charge between the phospholipids of the cytosolic and luminal leaflets of the bilayer (indicated by the yellow + and – signs) is also thought to play a role in determining the membrane protein topology. The transmembrane segments are shown as helices based on studies indicating that these regions adopt a helical secondary structure within the ribosomal exit tunnel before they enter the translocon. In recent years, new classes of transmembrane proteins have been found that bypass the translocon. One such class of proteins, called tail-anchored proteins, have extended N-terminal cytosolic domains and a single C-terminal membrane domain that also serves as a signal sequence. Since the signal sequence is at the C-terminus, it is the last segment of the protein to be translated by the ribosome, so tail-anchored proteins cannot be targeted for co- translational translocation. Yet, they still manage to be properly inserted into the ER membrane. After these proteins are synthesized in the cytoplasm, they are targeted to the ER through a series of interactions with proteins in the GET (Guided Entry of Tail-Anchored proteins) pathway. Recent research has elucidated the basic steps of the GET pathway. Tail-anchored proteins in the cytosol are first captured by a cytosolic protein and then transferred to an ER-directed “targeting complex” of Get proteins (in yeast, made up of Get3, Get4, and Get5). At the ER membrane, a transmembrane protein complex (composed of Get1 and Get2) binds to the tail- anchored protein and inserts it into the ER membrane. It is estimated that there are hundreds of tail-anchored proteins in eukaryotes that must utilize the GET pathway or alternative pathways in order to be properly localized to the ER membrane. Membrane Biosynthesis in the Endoplasmic Reticulum Membranes do not arise de novo, that is, as new entities from pools of protein and lipid that mix together. Instead, membranes arise from preexisting membranes. Membranes grow as newly synthesized proteins, and lipids are inserted into existing membranes in the endoplasmic reticulum (ER). As will become apparent in the following discussion, membrane components move from the ER to virtually every other compartment in the cell. As the membrane moves from one compartment to the next, its proteins and lipids are modified by enzymes that reside in the cell’s various organelles. These modifications contribute to giving each membrane compartment a unique composition and distinct identity. Keep in mind that cellular membranes are asymmetric: The two phospholipid layers (leaflets) of a membrane have different compositions (Section 4.2). This asymmetry is established initially in the ER. Asymmetry is maintained as membrane carriers bud from one compartment and fuse to the next. As a result, domains situated at the cytosolic surface of the ER membrane can be identified on the cytosolic surface of transport vesicles, the cytosolic surface of Golgi cisternae, and the internal (cytoplasmic) surface of the plasma membrane (Figure 8.15). Similarly, domains situated at the luminal surface of the ER membrane maintain their orientation and are found at the external (exoplasmic) surface of the plasma membrane. In fact, in many ways, including its high calcium concentration and abundance of proteins with disulfide bonds and carbohydrate chains, the lumen of the ER (along with other compartments of the secretory pathway) is a lot like the extracellular space. FIGURE 8.15 Maintenance of membrane asymmetry. As each protein is synthesized in the RER, it becomes inserted into the lipid bilayer in a predictable orientation determined by its amino acid sequence. This orientation is maintained throughout its travels in the endomembrane system, as illustrated in this figure. The carbohydrate chains, which are first added in the ER, provide a convenient way to assess membrane sidedness because they are always present on the cisternal side of the cytoplasmic membranes, which becomes the exoplasmic side of the plasma membrane following the fusion of vesicles with the plasma membrane. Most membrane lipids are synthesized entirely within the ER. The major exceptions are (1) sphingomyelin and glycolipids, synthesis of which begins in the ER and is completed in the Golgi complex, and (2) some of the unique lipids of the mitochondrial and chloroplast membranes, which are synthesized by enzymes that reside in those membranes. The enzymes involved in the synthesis of phospholipids are themselves integral proteins of the ER membrane with their active sites facing the cytosol. Newly synthesized phospholipids are inserted into the half of the bilayer facing the cytosol. Some of these lipid molecules are later flipped into the opposite leaflet through the action of enzymes called flippases. Lipids are carried from the ER to the Golgi complex and plasma membrane as part of the bilayer that makes up the walls of transport vesicles. The membranes of different organelles have markedly different lipid composition (Figure 8.16a), which indicates that changes take place as membrane flows through the cell. Several factors may contribute to such changes (Figure 8.16b): 1. Most membranous organelles contain enzymes that modify lipids already present within a membrane, converting one type of phospholipid (e.g., phosphatidylserine) to another (e.g., phosphatidylcholine) (step 1, Figure 8.16b). 2. When vesicles bud from a compartment (as in Figure 8.2a), some types of phospholipids may be preferentially included within the membrane of the forming vesicle, while other types may be left behind (step 2, Figure 8.16b). 3. In many microscopy studies focusing on the ER, segments of the ER have been observed to form direct and dynamic contacts with other organelles, including the Golgi complex, mitochondria, and plasma membrane. Recent studies have shown that these membrane contact sites represent regions where two intracellular compartments are tethered together in close proximity (within 30 nm) without complete fusion of the membrane bilayers. One function of these sites is to exchange lipids between the two compartments. Lipid exchange can be facilitated by lipid transfer proteins (step 3, Figure 8.16b), which may be soluble or tethered to membranes. Lipid transfer may also occur through the formation of a hydrophobic protein channel between the compartments. FIGURE 8.16 Modifying the lipid composition of membranes. (a) Histogram indicating the percentage of each of three phospholipids (phosphatidylcholine, phosphatidylserine, and sphingomyelin) in three different cellular membranes (ER, Golgi complex, and plasma membrane). The percentage of each lipid changes gradually as the membrane flows from the ER to the Golgi complex to the plasma membrane. (b) Schematic diagram showing three distinct mechanisms that might explain how the phospholipid composition of one membrane in the endomembrane system can be different from another, even though the membranous compartments are spatially and temporally continuous. (1) The head groups of phospholipids of the bilayer are modified enzymatically; (2) the membrane of a forming vesicle contains a different phospholipid composition than the membrane from which it buds; and (3) lipids can be removed from one membrane and inserted into another membrane by lipid transfer proteins. Glycosylation in the Rough Endoplasmic Reticulum Nearly all of the proteins produced on membrane-bound ribosomes—whether integral components of a membrane, soluble lysosomal or vacuolar enzymes, or parts of the extracellular matrix—become glycoproteins. Carbohydrate groups have key roles in the function of many glycoproteins, particularly as binding sites in their interactions with other macromolecules, which occurs during many cellular processes. They also aid in the proper folding and stabilization of the protein to which they are attached. The sequences of sugars that comprise the oligosaccharides of glycoproteins are highly specific; if the oligosaccharides are isolated from a purified protein of a given type of cell, their sequence is consistent and predictable. How is the order of sugars in oligosaccharides achieved? The addition of sugars to an oligosaccharide chain is catalyzed by a large family of membrane-bound enzymes called glycosyltransferases. Each of these enzymes transfers a specific monosaccharide from a nucleotide sugar, such as GDP-mannose or UDP- N-acetylglucosamine (Figure 8.17), to the growing end of the carbohydrate chain. The sequence in which sugars are transferred depends on the sequence of action of glycosyltransferases that participate in the process. This in turn depends on the location of specific enzymes within the various membranes of the secretory pathway. Thus, the arrangement of sugars in the oligosaccharide chains of a glycoprotein depends on the spatial localization of particular enzymes in the assembly line. Source: Adapted from D. Voet and J. G. Voet, Biochemistry, 2e, Copyright 1995; John Wiley & Sons, Inc. FIGURE 8.17 Steps in the synthesis of the core portion of an N-linked oligosaccharide in the RER. The first seven sugars (five mannose and two NAG residues) are transferred one at a time to the dolichol pyrophosphate (dolichol-PP) on the cytosolic side of the ER membrane (steps 1 and 2). At this stage, the dolichol with its attached oligosaccharide is then flipped across the membrane (step 3), and the remaining sugars (four mannose and three glucose residues) are attached on the luminal side of the membrane. These latter sugars are attached one at a time on the cytosolic side of the membrane to the end of a dolichol phosphate molecule (as in steps 4 and 7), which then flips across the membrane (steps 5 and 8) and donates its sugar to the growing end of the oligosaccharide chain (steps 6 and 9). Once the oligosaccharide is completely assembled, it is transferred enzymatically to an asparagine residue (within the sequence Asn-X-Ser/Thr) of the nascent polypeptide (step 10). The dolichol-PP is flipped back across the membrane (step 11) and is ready to begin accepting sugars again (steps 12 and 13). The initial steps in the assembly of N-linked oligosaccharides (as opposed to O-linked oligosaccharides; see Figure 4.11) of both soluble proteins and integral membrane proteins are shown in Figure 8.17. The basal, or core, segment of each carbohydrate chain is not assembled on the protein itself but put together independently on a lipid carrier and then transferred, as a block, to specific asparagine residues of the polypeptide. This lipid carrier, which is named dolichol phosphate, is embedded in the ER membrane. Sugars are added to the dolichol phosphate molecule one at a time by membrane-bound glycosyltransferases, beginning with step 1 of Figure 8.17. This part of the glycosylation process is essentially invariant; in mammalian cells, it begins with the transfer of N-acetylglucosamine 1-phosphate, followed by the transfer of another N-acetylglucosamine, then nine mannose and three glucose units in the precise pattern indicated in Figure 8.17. This preassembled block of 14 sugars is then transferred by the ER enzyme oligosaccharyltransferase from dolichol phosphate to certain asparagines in the nascent polypeptide (step 10, Figure 8.17) as the polypeptide is being translocated into the ER lumen. Mutations that lead to the total absence of N-glycosylation cause the death of embryos prior to implantation. However, mutations that lead to partial disruption of the glycosylation pathway in the ER are responsible for serious inherited disorders affecting nearly every organ system. These diseases are called congenital disorders of glycosylation, or CDGs, and are usually identified through blood tests that detect abnormal glycosylation of serum proteins. One of these diseases, CDG1b, can be managed through a remarkably simple treatment. CDG1b results from the deficiency of the enzyme phosphomannose isomerase, which catalyzes the conversion of fructose 6-phosphate to mannose 6-phosphate, a crucial reaction in the pathway that makes mannose available for incorporation into oligosaccharides. The disease can be managed by providing patients with oral supplements of mannose. The treatment was first tested in a boy who was dying from uncontrolled gastrointestinal bleeding, one of the usual complications of the disease. Within months of taking mannose supplements, the child was living a normal life. Shortly after it is transferred to the nascent polypeptide, the oligosaccharide chain undergoes a process of gradual modification. This modification begins in the ER with the enzymatic removal of two of the three terminal glucose residues (step 1, Figure 8.18). This sets the stage for an important event in the life of a newly synthesized glycoprotein. The glycoprotein is screened by a system of quality control that determines whether it is fit to move on to the next compartment of the biosynthetic pathway. To begin this screening process, each glycoprotein, which at this stage contains a single remaining glucose, binds to an ER chaperone (calnexin or calreticulin) (step 2). Removal of the remaining glucose by glucosidase II leads to the release of the glycoprotein from the chaperone (step 3). If a glycoprotein at this stage has not completed its folding or has misfolded, it is recognized by a conformation-sensing enzyme (called UGGT) that adds a single glucose residue back to one of the mannose residues at the exposed end of the recently trimmed oligosaccharide (step 4). UGGT recognizes incompletely folded or misfolded proteins because they display exposed hydrophobic residues that are absent from properly folded proteins. Once the glucose residue has been added, the “tagged” glycoprotein is recognized by the same ER chaperones, which give the protein another chance to fold properly (step 5). After a period of time with the chaperone, the added glucose residue is removed and the conformation-sensing enzyme checks it again to see if it has achieved its proper three-dimensional structure. If it is still partially unfolded or misfolded, another glucose residue is added and the process is repeated until, eventually, the glycoprotein has either folded correctly and continues on its way (step 6) or remains misfolded and is destroyed. Studies suggest that the “decision” to destroy the defective protein begins with the activity of a slow-acting enzyme in the ER. The enzyme trims a mannose residue from an exposed end of the oligosaccharide of a protein that has been in the ER for an extended period. Once one or more of these mannose residues have been removed (step 7), the protein can no longer be recycled and, instead, is sentenced to degradation (step 8). Source: Adapted from L. Ellgaard et al., Science 286:984, 1999; copyright 1999, from AAAS. FIGURE 8.18 Quality control: ensuring that misfolded proteins do not proceed forward. Based on this proposed mechanism, misfolded proteins are recognized by a glucosyltransferase (UGGT), which adds a glucose to the end of the oligosaccharide chains. Glycoproteins containing monoglucosylated oligosaccharides are recognized by the membrane-bound chaperone calnexin and given an opportunity to achieve their correctly folded (native) state. If that does not occur after repeated attempts, the protein is dislocated to the cytosol and destroyed. The steps are described in the text. A soluble chaperone (calreticulin) participates in this same quality control pathway. We will pick up the story of protein glycosylation again in Section 8.4 where the oligosaccharide that is assembled in the ER is enlarged as it passes through the Golgi complex on its journey through the biosynthetic pathway. Mechanisms That Ensure the Destruction of Misfolded Proteins You have just seen how proteins that fail to fold properly are detected by ER enzymes. It was a surprise to discover that misfolded proteins are not destroyed in the ER, but instead are transported into the cytosol. The precise mechanism of translocation across the ER membrane remains a matter of debate. Once in the cytosol, the oligosaccharide chains are removed, and the misfolded proteins are destroyed in proteasomes. The structure and function of these protein-degrading machines are discussed in Section 12.11. This process, known as ER-associated degradation (ERAD), ensures that aberrant proteins are not transported to other parts of the cell, but it can have negative consequences. Over 60 human diseases, including cystic fibrosis, are attributed to the ERAD pathway. In most patients with cystic fibrosis, the plasma membrane of epithelial cells is lacking the abnormal protein encoded by the cystic fibrosis gene (see the Human Perspective feature in Section 4.6). In these cases, the mutant protein (which often would be functional if allowed sufficient time to fold) is destroyed by the quality control process of the ER and thus fails to reach the cell surface. Under certain circumstances, misfolded proteins can be generated in the ER at a rate faster than they can be exported to the cytoplasm. The accumulation of misfolded proteins, which is potentially lethal to cells, triggers a comprehensive plan of action within the cell known as the unfolded protein response (UPR). The ER contains protein sensors that monitor the concentration of unfolded or misfolded proteins in the ER lumen. According to the prevailing model outlined in Figure 8.19, the sensors are normally kept in an inactive state by molecular chaperones, particularly BiP. If circumstances should lead to an accumulation of misfolded proteins, the BiP molecules in the ER lumen are called into service as chaperones for the misfolded proteins, rendering them incapable of inhibiting the sensors. Activation of the sensors leads to a multitude of signals that are transmitted into both the nucleus and the cytosol and result in: The expression of hundreds of different genes with encoded proteins that have the potential to alleviate stressful conditions within the ER. These include genes that encode (1) ER-based molecular chaperones that can help misfolded proteins reach the native state, (2) proteins involved in the transport of the proteins out of the ER, and (3) proteins involved in the selective destruction of abnormal proteins as discussed above. Phosphorylation of a key protein (eIFα) required for protein synthesis. This modification inhibits protein synthesis and decreases the flow of newly synthesized proteins into the ER. This gives the cell an opportunity to remove those proteins that are already present in the ER lumen. FIGURE 8.19 A model of the mammalian unfolded protein response (UPR). The ER contains transmembrane proteins that function as sensors of stressful events that occur within the ER lumen. Under normal conditions, these sensors are present in an inactive state as the result of their association with chaperones, particularly BiP (step 1). If the number of unfolded or misfolded proteins should increase to a high level, the chaperones are recruited to aid in protein folding, which leaves the sensors in their unbound, activated state and capable of initiating a UPR. At least three distinct UPR pathways have been identified in mammalian cells, each activated by a different protein sensor. Two of these pathways are depicted in this illustration. In one of these pathways, the release of the inhibitory BiP protein leads to the dimerization of a sensor (called PERK) (step 2). In its dimeric state, PERK becomes an activated protein kinase that phosphorylates a protein (eIF2α) required for the initiation of protein synthesis (step 3). This translation factor is inactive in the phosphorylated state, which stops the cell from synthesizing additional proteins in the ER (step 4), giving the cell more time to process those proteins already present in the ER lumen. In the second pathway depicted here, the release of the inhibitory BiP protein allows the sensor (called ATF6) to move on to the Golgi complex where the cytosolic domain of the protein is cleaved away from its transmembrane domain (step 2a). The cytosolic portion of the sensor diffuses through the cytosol (step 3a) and into the nucleus (step 4a), where it stimulates the expression of genes encoding proteins that can alleviate the stress in the ER (step 5a). These include chaperones, coat proteins that form on transport vesicles, and proteins of the quality control machinery. Interestingly, the UPR is more than a cell survival mechanism; it also includes the activation of a pathway that leads to the death of the cell. It is presumed that the UPR provides a mechanism to relieve itself of the stressful conditions. If these corrective measures are not successful, the cell death pathway is triggered and the cell is destroyed. ER to Golgi Vesicular Transport Membrane vesicles, with their enclosed cargo, bud from the edges of the ER and are targeted to the Golgi complex. The RER cisternae contain specialized exit sites devoid of ribosomes that serve as places where the first transport vesicles in the biosynthetic pathway are formed. The trip from the ER toward the Golgi complex can be followed visually in living cells by tagging secretory proteins with green fluorescent protein (GFP). Using this technique, it was found that, soon after they bud from the ER membrane, transport vesicles fuse with one another to form larger vesicles and interconnected tubules in the region between the ER and Golgi complex. This region has been named the ERGIC (endoplasmic reticulum Golgi intermediate compartment), and the vesicular–tubular carriers that form there are called VTCs (see Figure 8.26a). Once formed, the VTCs move farther away from the ER toward the Golgi complex. The movement of two of these vesicular–tubular membranous carriers from the ERGIC to the Golgi complex is shown in Figure 8.20. Movement of VTCs occurs along tracks composed of microtubules. Source: Reprinted by permission from Springer Nature: John F. Presley et al. Nature 389, pages 81–85, 1997. FIGURE 8.20 Visualizing membrane traffic with the use of a fluorescent tag. This series of photographs shows a small portion of a living mammalian cell that has been infected with the vesicular stomatitis virus (VSV) containing a VSVG–GFP chimeric gene (Figure 8.4). Once it is synthesized in the RER, the fusion protein emits a green fluorescence, which can be followed as the protein moves through the cell. In the series of photographs shown here, two vesicular–tubular carriers (VTCs) (arrows) containing the fluorescent protein have budded from the ER and are moving toward the Golgi complex (GC). The series of events depicted here took place over a period of 13 seconds. Bar represents 6 μm. Review 1. What are the major morphological differences between the RER and the SER? What are the major differences in their functions? 2. Describe the steps that occur between the time a ribosome attaches to a messenger RNA encoding a secretory protein and the time the protein leaves the RER. 3. How are newly synthesized integral proteins inserted into a membrane? 4. Describe some of the ways that membranous organelles can maintain their unique compositions despite the continuous traffic of membranes and materials moving through them. 5. Describe how membrane asymmetry is maintained as a membrane moves from the ER to the plasma membrane. 6. Describe the quality control steps that take place in the RER to ensure that glycoproteins are properly folded. 7. Describe the mechanisms by which the cell ensures that misfolded proteins (a) will not go unrecognized within the ER and (b) will not accumulate to excessive levels within the ER lumen. 8.4 The Golgi Complex In the latter years of the nineteenth century, an Italian biologist, Camillo Golgi, was inventing new types of staining procedures that might reveal the organization of nerve cells within the central nervous system. In 1898, Golgi applied a metallic stain to nerve cells from the cerebellum and discovered a darkly stained reticular network located near the cell nucleus. This network, which was later identified in other cell types and named the Golgi complex, helped earn its discoverer the Nobel Prize in 1906. The Golgi complex remained a center of controversy for decades between those who believed that the organelle existed in living cells and those who believed it was an artifact, that is, an artificial structure formed during preparation for microscopy. It was not until the Golgi complex was clearly identified in unfixed, freeze-fractured cells (see Figure 18.20) that its existence was verified beyond reasonable doubt. The Golgi complex has a characteristic morphology consisting primarily of flattened, disklike, membranous cisternae with dilated rims and associated vesicles and tubules (Figure 8.21a). The cisternae, with diameters typically of 0.5–1.0 μm, are arranged in an orderly stack, much like a stack of pancakes, and are curved so as to resemble a shallow bowl (Figure 8.21b).3 Typically, a Golgi stack contains fewer than eight cisternae. An individual cell may contain from a few to several thousand distinct stacks, depending on the cell type. The Golgi stacks in mammalian cells form a single, large complex (Figure 8.21c) typically situated adjacent to the cell’s nucleus (Figure 8.21d). A closer look at an individual cisterna suggests that vesicles bud from a peripheral tubular domain of each cisterna (Figure 8.21e). As discussed later, many of these vesicles contain a distinct protein coat that is visible in Figure 8.21e. Source: (a) Adapted from A. Rambourg and Y. Clermont, copyright 1990, Eur J Cell Biol. Originally published in The Journal of Cell Biology, Volume 51:195; (b) Courtesy of Thomas H. Giddings and Andrew Staehelin; (c) ©2009 Scott Emr et al. Originally published in The Journal of Cell Biology. https://doi.org/10.1083/jcb.200909011; (d) ©2002 Andrei V. Nikonov. et al. Originally published in The Journal of Cell Biology. https://doi.org/10.1083/jcb.200201116; (e) From Peggy J. Weidman and John Heuser, Trends Cell Biol. 5:303, 1995; with permission from Elsevier. FIGURE 8.21 The Golgi complex. (a) Schematic model of a portion of a Golgi complex from an epithelial cell of the male rat reproductive tract. The elements of the cis and trans compartments are often discontinuous and appear as tubular networks. (b) Electron micrograph of a portion of a tobacco root cap cell showing the cis-to-trans polarity of the Golgi stack. (c) Electron tomographic image of a slice from a mouse pancreatic beta cell that synthesizes and secretes the protein insulin. The individual Golgi stacks are seen to be interconnected to form a continuous ribbon. The trans face (or TGN) of each Golgi stack has been colored red, and the cis face has been colored light blue. (d) Fluorescence micrograph of a cultured mammalian cell. The position of the Golgi complex is revealed by the red fluorescence, which marks the localization of antibodies to a COPI coat protein. (e) Electron micrograph of a single isolated Golgi cisterna showing two distinct domains: a concave central domain and an irregular peripheral domain. The peripheral domain consists of a tubular network from which protein-coated buds are being pinched off. The Golgi complex is divided into several functionally distinct compartments arranged along an axis from the cis or entry face closest to the ER to the trans or exit face at the opposite end of the stack (Figures 8.21a,b). The cis-most face of the organelle is composed of an interconnected network of tubules referred to as the cis Golgi network (CGN). The CGN is thought to function primarily as a sorting station that distinguishes between proteins to be shipped back to the ER (discussed further in Section 8.5) and those that are allowed to proceed to the next Golgi station. The bulk of the Golgi complex consists of a series of large, flattened cisternae, which are divided into cis, medial, and trans cisternae (Figure 8.21a). The trans-most face of the organelle contains a distinct network of tubules and vesicles called the trans Golgi network (TGN). The TGN is a sorting station where proteins are segregated into different types of vesicles heading either to the plasma membrane or to various intracellular destinations. The membranous elements of the Golgi complex are thought to be supported mechanically by a peripheral membrane skeleton or scaffold composed of a variety of proteins, including members of the spectrin, ankyrin, and actin families—proteins that are also present as part of the plasma membrane skeleton (Section 4.5). The Golgi scaffold may be physically linked with motor proteins that direct the movement of vesicles and tubules entering and exiting the Golgi complex. A separate group of membrane-associated proteins are thought to form a Golgi “matrix,” which plays a key role in the disassembly and reassembly of the Golgi complex during mitosis. Figure 8.22 provides visual evidence that the Golgi complex is not uniform in composition from one end to the other. Differences in composition of the membrane compartments from the cis to the trans face reflect the fact that the Golgi complex is primarily a “processing plant.” Newly synthesized membrane proteins, as well as secretory and lysosomal proteins, leave the ER and enter the Golgi complex at its cis face and then pass across the stack to the trans face. As they progress along the stack, proteins that were originally synthesized in the RER are sequentially modified in specific ways. In the best-studied Golgi activity, a protein’s carbohydrates are modified by a series of stepwise enzymatic reactions, as discussed in the following section. Source: (a) ©1974 Robert S. Decker, Originally published in The Journal of Cell Biology. https://doi.org/10.1083/jcb.61.3.599; (b) ©1993 Angel Velasco et al. Originally published in The Journal of Cell Biology. http://doi.org/10.1083/jcb.122.1.39; (c) ©1974 Robert S. Decker, Originally published in The Journal of Cell Biology. https://doi.org/10.1083/jcb.61.3.599 FIGURE 8.22 Regional differences in membrane composition across the Golgi stack. (a) Reduced osmium tetroxide preferentially impregnates the cis cisternae of the Golgi complex. (b) The enzyme mannosidase II, which is involved in trimming the mannose residues from the core oligosaccharide as described in the text, is preferentially localized in the medial cisternae. (c) The enzyme nucleoside diphosphatase, which splits dinucleotides (e.g., UDP) after they have donated their sugar, is preferentially localized in the trans cisternae. Glycosylation in the Golgi Complex The Golgi complex plays a key role in the assembly of the carbohydrate component of glycoproteins and glycolipids. When we left the topic of synthesis of N-linked carbohydrate chains, the glucose residues had just been removed from the ends of the core oligosaccharide. As newly synthesized soluble and membrane glycoproteins pass through the cis and medial cisternae of the Golgi stack, most of the mannose residues are also removed from the core oligosaccharides, and other sugars are added sequentially by various glycosyltransferases. In the Golgi complex, as in the RER, the sequence in which sugars are incorporated into oligosaccharides is determined by the spatial arrangement of the specific glycosyltransferases that come into contact with the newly synthesized protein as it moves through the Golgi stack. The enzyme sialyltransferase, for example, which places a sialic acid at the terminal position of the chain in animal cells, is localized in the trans face of the Golgi stack, as would be expected if newly synthesized glycoproteins were continually moving toward this part of the organelle. In contrast to the glycosylation events that occur in the ER, which assemble a single core oligosaccharide, the glycosylation steps in the Golgi complex can be quite varied, producing carbohydrate domains of remarkable sequence diversity. One of many possible glycosylation pathways is shown in Figure 8.23. Unlike synthesis of N-linked oligosaccharides, which begins in the ER, those attached to proteins by O-linkages (Figure 4.11) are assembled entirely within the Golgi complex. FIGURE 8.23 Steps in the glycosylation of a typical mammalian N-linked oligosaccharide in the Golgi complex. Following the removal of the three glucose residues, various mannose residues are subsequently removed, while a variety of sugars (N-acetylglucosamine, galactose, fucose, and sialic acid) are added to the oligosaccharide by specific glycosyltransferases. These enzymes are integral membrane proteins with active sites that face the lumen of the Golgi cisternae. This is only one of numerous glycosylation pathways. The Golgi complex is also the site of synthesis of most of a cell’s complex polysaccharides, including the glycosaminoglycan chains of the proteoglycan shown in Figure 7.9a and the pectins and hemicellulose found in the cell walls of plants (see Figure 7.35c). The Movement of Materials through the Golgi Complex That materials move through the various compartments of the Golgi complex has long been established; however, two contrasting views of the way this occurs have dominated the field for years. Up until the mid-1980s, it was generally accepted that Golgi cisternae were transient structures. It was supposed that Golgi cisternae formed at the cis face of the stack by fusion of membranous carriers from the ER and ERGIC and that each cisterna physically moved from the cis to the trans end of the stack, changing in composition as it progressed. This is known as the cisternal maturation model because each cisterna “matures” into the next cisterna along the stack. Between the mid-1980s and the mid-1990s, the maturation model of Golgi movement was largely abandoned and replaced by an alternate model, which proposed that the cisternae of a Golgi stack remain in place as stable compartments. In this latter model, which is known as the vesicular transport model, cargo (i.e., secretory, lysosomal, and membrane proteins) is shuttled through the Golgi stack, from the CGN to the TGN, in vesicles that bud from one membrane compartment and fuse with a neighboring compartment farther along the stack. The vesicular transport model is illustrated in Figure 8.24a, and its acceptance was based largely on the following observations: 1. Each of the various Golgi cisternae of a stack has a distinct population of resident enzymes (Figure 8.22). How could the various cisternae have such different properties if each cisterna was giving rise to the next one in line, as suggested by the cisternal maturation model? 2. Large numbers of vesicles can be seen in electron micrographs to bud from the rims of Golgi cisternae. In 1983, James Rothman and his colleagues at Stanford University demonstrated, using cell-free preparations of Golgi membranes (Section 8.2), that transport vesicles were capable of budding from one Golgi cisterna and fusing with another Golgi cisterna in vitro. This landmark experiment formed the basis for a hypothesis suggesting that inside the cell, cargo-bearing vesicles budded from cis cisternae and fused with cisternae situated at a more trans position in the stack. Source: (a) Adapted from Vesicular transport model. From Alexander A. Mironov et al., courtesy of Alberto Luini, J. Cell Biol. 155:1234, 2001 from The Rockefeller University Press; (b) Adapted from Cisternal maturation model. From Jose A. Martinez-menárguez et al., courtesy of Judith Klumperman, J. Cell Biol. 155:1214, 2001 from The Rockefeller University Press.; (c) ©2001 Alexander A. Mironov.et.al. Originally published in The Journal of Cell Biology. https://doi.org /10.1083/jcb.200108073; (d) ©2001 Judith Klumperman et al. Originally published in The Journal of Cell Biology. https://doi.org/10.1083/jcb.200108029. FIGURE 8.24 The dynamics of transport through the Golgi complex. (a) In the vesicular transport model, cargo (black dots) is carried in an anterograde direction by transport vesicles, while the cisternae themsel

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