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In the next two tutorials, we will discuss several of the intracellular compartments of a typical eukaryotic cell; specifically, the endomembrane system, which is composed of the endoplasmic reticulum, the Golgi complex, endosomes and lysosomes. You will learn about the functions of these organelles, and about the movement of molecules between them and the plasma membrane. In this tutorial we will consider the role of the endoplasmic reticulum and the Golgi complex. The flow of proteins between these two membrane networks will be examined, and the molecular mechanisms that guide this transport will be described.
By the end of this tutorial you should know:
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The endomembrane system , illustrated in Figure 1, is composed of the outer membrane of the nucleus, the endoplasmic reticulum, the Golgi complex, endosomes and lysosomes. Material can move freely between the outer membrane of the nucleus and the endoplasmic reticulum. The movement of material between the other components of the endomembrane system is mediated by vesicles , small regions of membrane that are pinched off from an organelle membrane. The vesicles migrate and fuse with another membrane. There is a great deal of trafficking of lipids and proteins between the endoplasmic reticulum, the Golgi complex, enodosomes, lysosomes and the plasma membrane. For example, secretory proteins, such as the peptide hormone insulin, are synthesized in association with the endoplasmic reticulum, transported to the Golgi complex, and finally, transported to the plasma membrane for secretion. The endoplasmic reticulum is also the site of lipid synthesis.
The endoplasmic reticulum (ER) is a network of flattened sacs and tubules in the cytoplasm of a cell. The term cisternae refers to the individual sacs, and the term lumen refers to the space enclosed by the membrane of the sacs and tubules. There are two distinct regions of the ER: the rough ER and smooth ER . The association of ribosomes with the outer surface of the ER membrane characterizes the rough ER. The smooth ER is characterized by the absence of ribosomes. Ribosomes, described in a previous tutorial, are the site of protein synthesis. The rough ER is the site of synthesis and processing of proteins destined for secretion or the endomembrane system. The smooth ER is predominantly the site of lipid synthesis, drug detoxification, catabolism of stored glycogen and calcium storage.
The smooth ER has several important metabolic roles, including lipid synthesis. Most membrane lipids are synthesized in the smooth ER. Newly synthesized lipids are incorporated into the outer (or cytoplasmic) side of the ER lipid bilayer. Specific phospholipid exchange proteins catalyze the flip-flop of lipids to the inner (or lumenal) side of the lipid bilayer. The smooth ER is also the site of drug detoxification. In liver cells, enzymes in the smooth ER carry out hydroxylation reactions (attachment of a hydroxyl group to an organic molecule), which increase the solubility of foreign compounds and facilitate their transport out of the cell, and subsequently, out of the body. In addition, the smooth ER in liver cells is involved in the break down of glycogen to release glucose. In many cells, the smooth ER is also the site of calcium storage. The lumen of the ER contains high concentrations of calcium. The ER membrane contains proteins that pump calcium into the lumen, as well as proteins that allow the release of calcium into the cytoplasm in response to intracellular signals.
Proteins that become part of the endomembrane system, the plasma membrane or that are secreted out of the cell are synthesized on the rough ER. (Recall, other proteins are synthesized on ribosomes found free in the cytosol of the cell.) Ribosomes are associated with the ER membrane, and while a protein is being translated it is inserted into the ER membrane. When translation is complete, the newly synthesized protein will remain in the ER membrane if it is a membrane protein, or it will be released into the lumen of the ER if it is a secretory or soluble protein. The mechanisms for directing a ribosome to the ER and the insertion of a growing polypeptide chain into the membrane were discussed in the tutorial describing protein translation. Enzymes in the ER modify and process proteins by cotranslational import (membrane insertion is coupled directly to translation) as polypeptides are being synthesized. As is illustrated in Figure 2, the nascent polypeptide begins to fold, facilitated by a chaperone protein that mediates protein folding and senses the correct conformation. Commonly, proteins synthesized in the rough ER are glycosylated (have short polysaccharides covalently attached). That is, enzymes in the ER add core oligosaccharides (a short-chain polysaccharide with between 4 and 20 monosaccharides ) to the nascent polypeptide. In addition, many secreted proteins have disulfide bonds, the formation of which is catalyzed by enzymes in the ER. Finally, if a protein is multimeric (consisting of two or more polypeptides), the subunits are assembled in the ER. Once a protein has been folded and modified in the ER, it is transported to the Golgi complex for further modification and sorting to the correct subcellular location.
The rough ER functions as the quality control center for newly synthesized proteins, ensuring that only correctly folded and correctly assembled proteins are transported to the Golgi complex. Any misfolded polypeptides or incorrectly assembled multimeric proteins are transported out of the ER and targeted for degradation by a protein complex in the cytoplasm referred to as a proteasome . In response to the accumulation of misfolded proteins, or even large amounts of normal proteins in the ER, the cell will trigger the unfolded protein response . This pathway results in the increased expression of the genes that encode all aspects of ER function. The unfolded protein response is tightly regulated and is the cell's mechanism of responding to and alleviating the accumulation of proteins in the ER.
There are two distinct types of protein glycosylation that occur in the rough ER: N-linked glycosylation , which involves the addition of an oligosaccharide to the amino group (NH2 ) of asparagine; and O-linked glycosylation , which involves the addition of an oligosaccharide to the hydroxyl group (OH) of serine and threonine. The initial addition of the oligosaccharide occurs in the ER, and then, it is further modified in the ER and Golgi complex. N-linked glycosylation occurs simultaneously with translation (illustrated in Figure 3). A core oligosaccharide, consisting of fourteen monosaccharides, is assembled and attached to a lipid carrier on the outer side of the lipid bilayer, which is then translocated across the ER membrane and added to asparagine residues of a nascent polypeptide. After the addition of the core oligosaccharide, sugars are cleaved in the ER, and then, in some instances, further modified in the Golgi complex. O-linked glycosylation occurs through the stepwise addition of monosaccharides in either the ER or Golgi complex. Most O-linked oligosaccharides are short, containing only four sugars.
The Golgi complex is illustrated in Figure 4. It is a series of stacked, flattened discs (also termed cisternae, like those of the ER) surrounded by numerous membrane vesicles. The Golgi complex has distinct regions that perform distinct activities. The cis face receives vesicles from the rough ER, and the trans face ships vesicles to the plasma membrane or endosomes. Proteins enter the Golgi complex and move through the cisternae. Movement is mediated by vesicles forming and fusing between cisternae in the Golgi complex, or by the maturation of cisternae from cis to trans regions. Movement of vesicles can be anterograde , moving from cis to trans, or retrograde , moving from trans to cis.
The primary roles of the Golgi complex include further modification of glycosylated proteins and the sorting of proteins to their appropriate subcellular domain. N-linked glycosylation proteins are further remodeled in the Golgi complex. The oligosaccharides may be cleaved, added, or modified by phosphorylation. O-linked glycosylation continues in the Golgi complex. In addition, some secretory proteins are cleaved to complete their maturation within, or as they leave, the Golgi complex for secretory vesicles. The enzymes that catalyze these reactions are compartmentalized in different regions of the Golgi complex. The complete processing of a protein occurs in a stepwise fashion, moving from the cis face to the trans face of the Golgi complex.
Most proteins synthesized in the rough ER are transported to the Golgi complex, and from there they are transported to their final destination. This includes transport from the ER to the Golgi complex and back to the ER; transport from the ER to the Golgi complex, and then to secretory vesicles that deliver proteins to the plasma membrane; and transport from the ER to the Golgi complex, and then to endosomes (which mature into lysosomes). The multiple sorting pathways are illustrated in Figure 5.
Proteins destined for the endomembrane system, the plasma membrane or secretion are synthesized in the rough ER, and then transported to the Golgi complex. It is in the Golgi complex that they are sorted to the correct subcellular location. This sorting is achieved by the presence of "tags" associated with the proteins themselves, which are recognized by the Golgi complex and used to direct the proteins to the correct organelle. The sorting tags vary for different types of proteins and include amino acid sequences, particular sugar residues, and general hydrophobicity and size of a protein. Next we will consider the mechanism of protein targeting and the retention of various types of proteins secreted or associated with the endomembrane system.
In the Golgi complex, proteins are sorted into secretory vesicles that bud off from the trans face, and they eventually fuse with the plasma membrane to deliver their contents. Many different secretory proteins are packaged into each secretory vesicle. Membrane proteins that are destined for microdomains of the plasma membrane, so-called lipid rafts (see tutorial on Membrane Structure and Function), will be packaged together and delivered to the plasma membrane. There are two classes of secretory vesicles: constitutive secretory vesicles, which fuse with the plasma membrane continuously; and regulated secretory vesicles, which are stored in the cell until they are signalled to fuse with the plasma membrane. For example, regulated secretion occurs in the specialized cells of the pancreas that synthesize insulin. Insulin is processed in the Golgi complex and packaged into secretory vesicles that are only released when the cells receive the signal that there is elevated blood glucose.
Most ER proteins are transported to the Golgi complex after synthesis, then retrieved; however, some ER proteins never leave the ER. The mechanism of ER retention is not clear, but may include aggregation of ER proteins so that they are not packaged into transport vesicles headed for the Golgi complex. Most soluble proteins present in the ER lumen are transported to the Golgi complex after synthesis, then retrieved. This retrieval is mediated by an amino acid sequence (lysine-aspartic acid-glutamatic acid-leucine) in the C-terminus of soluble ER proteins. Specific receptors in the ER and Golgi complex membranes recognize and bind this sequence. The receptor-protein complex is either retained in the ER or, more often, transported from the Golgi complex back to the ER via retrograde transportation (see Figure 6).
Golgi complex proteins are also synthesized in the rough ER and transported to the Golgi complex, where they remain. This retention is not mediated by a specific amino acid tag, but rather, through a structural feature, a hydrophobic transmembrane alpha helix. Interestingly, the length of the transmembrane domain may determine in which compartment within the Golgi complex they remain; the membrane of the trans face is thicker than the cis face, and proteins associated with that membrane have longer transmembrane domains.
Lysosomal proteins are synthesized in the rough ER and transported through, and modified in, the Golgi complex before being delivered to the endosomes, which mature into lysosomes. The lysosomal hydrolases (enzymes in the lumen of the lysosome that function to break down macromolecules) are sorted in the Golgi complex based on the presence of a specific sugar residue, mannose-6-phosphate . This is illustrated in Figure 7. Lysosomal hydrolases are recognized in the Golgi complex by the enzyme N-acetylglucosamine (GlcNac) phosphotransferase, which adds a mannose-6-phosphate to an N-linked oligosaccharide. The modified lysosomal proteins are then recognized and bound by the mannose-6-phosphate receptor (M6P receptor) located in the membrane of the Golgi complex. Transport vesicles are formed at the trans face of the Golgi complex containing the M6P receptor bound to the lysosomal proteins. These vesicles are destined to fuse with endosomes. The low pH in the endosomes triggers the dissociation of the M6P receptor-protein complex and the activation of the lysosomal hydrolases, resulting in a mature lysosome.
The endomembrane system includes the ER and the Golgi complex. The ER has two distinct domains: smooth ER and rough ER (distinguished by ribosomes associated with its membrane). The functions of the smooth ER include lipid synthesis, drug detoxification, glycogen catabolism and calcium storage. The main function of the rough ER is protein synthesis of the proteins that will be inserted into the plasma membrane, secreted, or associated with the endomembrane system. The rough ER is also the site of the correct folding and assembly of newly synthesized proteins, formation of disulfide bonds, and initiation of glycosylation. There are two types of protein glycosylation: N-linked and O-linked; both link oligosaccharides to different amino acids via distinct mechanisms. Most of the proteins synthesized in the rough ER will be transported to the Golgi complex, then sorted to the correct subcellular location. Aberrantly folded proteins are recognized as such and not transported to the Golgi complex; rather, they are targeted for degradation by the proteasome.
The Golgi complex is the site of modification of the core oligosaccharides added in the ER, including addition, removal and phosphorylation of oligosaccharides. The Golgi complex also is the site of protein sorting. Proteins enter the Golgi complex from the ER and can follow several pathways, including retention in the Golgi complex, retrieval from the Golgi complex to the ER, transport from the Golgi complex to the plasma membrane, or transport from the Golgi complex to the endosomes. Movement of proteins is mediated by vesicle transport in the anterograde and retrograde fashions. There are several distinct tags used to sort proteins to the correct organelle, including an amino acid tag for the retention and retrieval of soluble ER proteins, a structural feature of the transmembrane domain for the retention of Golgi complex membrane proteins, and an oligosaccharide tag for sorting lysosomal hydrolases.