Chapter 10.

Endoplasmic Reticulum
10.1 Morphology, Ultrastructure and Chemical Composition In different types of cells several different structures were observed in light microscopy by specific staining methods: Nissls bodies as dense spots in neurons, Bergs bodies in hepatocytes, or ergastoplasm basophilic regions in exocrine cells in pancreas. Using the electron microscope, K. Porter described in 1945 a system of intracellular membranes, called by him endoplasmic reticulum (ER), proved later to be a common cellular organelle for all eukaryotes. ER consists of flattened cisternae and interconnected tubes; it continues the outer nuclear membrane and extends within the entire cytoplasm without coming in contact with plasmalemma. G.E. Palade discovered ribonucleoproteic granules on the surface of the ER membranes, later called ribosomes. He also described their role in protein synthesis. Presence of these granules divided the ER in two forms: rough ER (RER), with ribosomes attached, and smooth ER (SER) without ribosomes. Between RER and SER there is no border: the lumen and the membranes of the two forms are continuous. Chemical composition of ER was studied after cell fractioning by differential centrifugation. During homogenization process required by this technique, the ER network of membranes is disrupted in small vesicles called microsomes. Therefore the microsomes are not cellular organelles. They are of two types, rough and smooth microsomes, according to the presence or absence of ribosomes on their membranes. Proteins : lipids ratio in the ER membrane is 1:1. The proteins may have sugar groups resulting in glycoproteins which are asymmetric molecules, displaying the sugar groups on the opposite face to cytoplasm. The majority of lipids are phospholipids: lecithin 45%, kephalin 30%, phosphatydil-serine 10% and phosphatydil-inosytol 5%, with very un-saturated fatty acids. The ER membranes also have a low amount of cholesterol that taken together with the un-saturated fatty acids confers a high fluidity to the ER membrane. 10.2 Specific Functions of RER Essential function of RER is protein synthesis, performed by the attached ribosomes. This is why the RER is well developed in cells with intense protein synthesis such as cells of the exocrine pancreas that produce daily kilograms of digestive enzymes, in neurons that synthesize neurotransmitters and plasma membrane proteins in high amounts, or in hepatocytes that synthesize plasmatic proteins.
1

Ribosomes attached to the RER are responsible for synthesis of proteins for cellular secretion (export proteins) and proteins that remain included in compartments of secretion pathway: lumen of RER and SER, sacks of Golgi complex, membranes of secretion vesicles. They also synthesize lysosomal enzymes and membrane proteins as well. Other cellular proteins, for example soluble proteins from cytosol, cytoplasmic differentiations and nuclear matrix proteins are synthesized by free ribosomes in cytoplasm. Mechanism of protein synthesis Many ribosomes may be linked to the same mRNA molecule in order to synthesize the same protein. They form a functional group named polyribosome or shorter, polysome. The ribosome read the information encoded by mRNA and the new protein will always be synthesized from the amino-terminus (N-terminus) to the carboxyl-terminus (C-terminus). Synthesis of proteins starts in the ribosome before its attachment to the RER membrane. The first segment of amino acids enchained has a characteristic structure. It is known as signal sequence or insertion signal and consists of the first 18-20 amino acids that get out from ribosome. This fragment is followed by another segment of 50-70 amino acids remained inside the ribosome. In the Nterminus zone of the signal sequence there are positive-charged amino acids followed by a hydrophobic region with 6-12 amino acids with an -helix structure; the positive residues of amino acids can link to the negative-charged polar groups of phospholipids from the RER membrane and the hydrophobic residues can insert in the membrane. This structure is suitable for the insertion of proteins in membrane. There are several proteins involved in the attachment of ribosomes to the RER membrane (the so called docking process) and in the transfer of the growing polypeptide chain through the RER membrane during its synthesis (cotranslational transfer). The signal sequence attached to the ribosome is recognized by a ribonucleo-proteic complex called the signal recognition particle (SRP). The SRP consists of 6 polypeptides with MW of 10,000-70,000 Da and one RNA 7S molecule. The SRP linked to ribosome arrests the synthesis of the growing polypeptide chain until the ribosome will be attached to the RER membrane. Another protein located in the RER membrane participates to the attachment of the ribosome to membrane: a receptor for SRP which links the SRP. In the RER membrane under the ribosome a trans-membrane protein channel is assembled by lateral diffusion. At this point the protein synthesis is resumed. The SRP receptor separates from the SRP that is released and will be recycled (to link another ribosome), and the SRP receptor will diffuse through membrane to link another SRP attached to another ribosome. The growing polypeptide is translated through the membrane into the RER lumen
2

during its synthesis until the synthesis is completed. The signal sequence is cut off by a signal peptidase and remains inserted in membrane. As the polypeptide chain flows through the channel is folded in a three dimensional final conformation. This conformation is given according to a minimal thermodynamic energy depends on the amino acids sequence. The folding process polypeptide chain is achieved by specific proteins for each molecule, named binding proteins (BiP) or chaperons that dissociate after the final structure was accomplished. The entire process, called vectorial transfer of the polypeptide chain, is characteristic for secretion proteins and is achieved with minimal energy consumption. When the synthesis of polypeptide is finished all the vectorial transfer apparatus is disassembled and the ribosome dissociates into their sub-units. Synthesis of membrane proteins is performed through a different mechanism. The first steps are identical, but these proteins have hydrophobic sequences of 20-25 amino acids that induce the disassembly of the channel when passing through it. The channel dissociates and allows the chain to interact freely with the hydrophobic regions of the lipids. The hydrophobic sequences are followed by other hydrophilic sequences and the channels will be reassembled. This way the polypeptide chain may form loops through the RER and may pass until 12 times the membrane and the amino terminus may be on any part of membrane. During synthesis the proteins are further modified: - Glycosylation is a biochemical reaction by which sugar groups are attached to the growing polypeptide chain. An oligosaccharide chain is first assembled on a lipid molecule from the RER membrane named dolichol. The monosaccharides are carried by uridine-diphosphate (UDP), and after the oligosaccharide is completed it is transferred on certain amino acids, especially on asparagin by specific enzymes called glycosyltransferases. - Formation of bi-sulphidic bonds with important roles in the spatial structure of the proteins. - The lateral chains of amino acids are modified. - Proteolysis is a biochemical reaction through which the polypeptide chain is cut by proteases.

10.3 Specific Functions of SER The main function of SER is represented by synthesis of lipids. Therefore the SER is present in high amounts as interconnected tubes in cells with a high synthesis of lipids. It is well developed in liver while in other organs such as adrenal glands and gonads (that synthesize steroid hormones) is the prevalent organelle and is distributed within all the cytoplasm. In enterocytes
3

that have an important role in absorption, the SER is also present in high amounts. Fatty acids, monoglycerides and diglycerides resulted from digestion of fats are absorbed by enterocyte at the apical pole and the SER from this cell will use them in order to resynthesize triglycerides. The triglycerides arrive in blood as chylomicrons, giving the turbid aspect of plasma after rich meals. The SER has two specific functions in hepatocytes: degradation of glycogen and detoxification. Glucose residues are released from glycogen by glycogenolysis as glucose-1-phosphate which in cytosol is transformed in glucose-6-phosphate. The latter is dissociated by glucose-6phosphatase inside the SER, and the glucose is released in cytosol free to pass into blood. Glucose-6-phosphatase is a marker enzyme for SER. For detoxification the exogenous and endogenous toxic substances are processed by different reactions: oxidation, reduction, hydrolysis, or conjugation with glucuronic acid. The substrates become either more soluble in order to be easier eliminated through kidneys, or are inactivated so they lose their biological effect and their toxic properties. These reactions are involved in the detoxification of environmental pollutants, medicines, or substances normally produced in metabolism: gall salts, steroid hormones, cholesterol, and hemoglobin. Red blood cells have a life span of about 120 days. The old red blood cells are recognized by phagocytes especially in spleen and are phagocytosed. These phagocytes degrade hem from hemoglobin (in SER) and produces bilirubin which is released in blood (as indirect bilirubin, attached to albumin). The blood transports it to hepatocytes where is conjugated with the glucuronic acid (in SER) by glucuronyl-transferase and the indirect bilirubin becomes direct bilirubin, which is soluble and is secreted in gall bladder, and from here in intestine. In hepatitis and jaundice the direct bilirubin is increased in blood. Most of the detoxification reactions occur in liver as oxidations performed by a specific microsomal system of enzymes named microsomal mixed-function oxidases. These oyidases use NADPH and O2. The main enzyme is a cytochrome with an iron ion that oscillates between the electrical charged forms 3+ and 2+. Because this enzyme has maximal light absorption at wavelength of 450 nm, it was named cytochrome P450. It links both the substrate and the oxygen:

Hydrogen from the final HO group comes from substrate and the electrons are provided by a short electron transport chain.

10.4 Common Functions of RER and SER 1. Synthesis of phospholipids is performed by enzymes that are membrane proteins, with the active site on the cytosolic side of the ER membrane. Precursors of phospholipids (fatty acidCoA and glycerolphosphate) are taken from cytosol and the newly synthesized phospholipids are included in the cytosolic layer of the ER membrane. In order to prevent the asymmetric growth of the membrane some of these phospholipids are transferred to the inner layer of the membrane by a transverse diffusion named flip-flop performed by an enzyme named flipase. From the ER membrane, the phospholipids can be sent to the nuclear envelope and to membranes of all other cellular organelles. 2. The essential function of ER is biogenesis of membranes. In ER both membrane proteins and membrane lipids are synthesized. The majority of new membrane compounds reach over some organelles and plasma membrane by transport vesicles budded from ER. These vesicles fuse with other organelles like Golgi apparatus or with plasmalemma where the new membrane components are included. In mitochondria the new phospholipids are brought from the ER membrane by specialized transfer proteins that are soluble cytosolic proteins. They release the phospholipids in the outer mitochondrial membrane. Membrane proteins for mitochondria and peroxysomes are posttranslationally imported directly from cytosol. 3. The ER is a cytoplasmatic circulatory system for the intracellular transport of substances, on different ways: they can move through the lumen of ER or they can move within the membrane by lateral diffusion. Since the ER membrane is very fluid it allows the diffusion of proteins and lipids to the outer nuclear membrane, or to different regions of the ER itself. 4. Because the ER is in cytoplasm it is assumed that it has a role of mechanic support and of compartmentalization of cytoplasm. 5. The ER membrane mediates exchanges between cytosol and lumen of ER through passive and active transport. There are trans-membrane ionic gradients and membrane potential at this level. One of the important processes is the active transport of calcium by a Ca2+-ATPase inside the ER which is a cytoplasmic repository of Ca, and an important contributor to the optimal cytosolic calcium concentration.

10.5 Medical Implications of ER

Young and very active cells contain RER in high amounts. This is why the cytoplasm of such cells stains in dark blue with basic dyes (cytoplasmic basophilia). Since the cancer cells are very active and in their cytoplasm the RER prevails, this staining method is also used in the diagnosis of cancer. On the other hand, the old cells contain SER and therefore the cytoplasm is stained in pink-red with acidic dyes (cytoplasmic eosinophilia). The SER highly proliferates when there is a high need to metabolize toxic substances. It is also the case of some medicines metabolized at this level. Phenobarbital, a drug used in therapy of epilepsy induces the proliferation of SER. In laboratory animals it was observed that Phenobarbital doubles the amount of SER in several days. Induction of microsomal enzymes is important in practice. If two SER metabolized medicines are given together, they will inhibit reciprocally their metabolism. Chronically ingested ethanol also induces the microsomal enzymes and accelerates the metabolism of medicines and therefore their doses need to be adjusted. Acute ingestion of ethanol inhibits the metabolism of medicines. Some carcinogenic agents induce cytochrome P448, which activates the carcinogenesis; thus a protection mechanism against toxic compounds can turn against the organism. High doses of toxic substances result in ER lesions and alterations; these alterations allow the diagnostic of intoxication in legal medicine. In well fed laboratory animals important amounts of RER were observed while after starvation RER was present in small amounts or was absent.

Chapter 11. Golgi apparatus

11.1. Morphology and ultrastructure

Camillo Golgi discovered Golgi apparatus by neurons impregnation with silver nitrate. He named it internal reticular apparatus, but its existence generated a controversy of 50 years, because it was believed that is only an artifact produced by the silver nitrate. On the electron microscopy photographs the Golgi apparatus is observed as a system of membranes and vesicles placed in the proximity of nucleus. Actually it consists of three components. The essential part is represented by 5-11 flattened sacks. In most of the cases the sacks are curved, with the convex region (proximal) oriented towards nucleus and the concave one (distal) towards plasmalemma. The Golgi sacks are divided in three regions: the sacks of the cis face (on the proximal face - convex), the middle sacks, and the sacks on the opposite face (distal) trans face. The second part of the Golgi apparatus is represented by small vesicles located between the ER and Golgi apparatus. They separate from the ER membranes and fuse with sacks on the Golgi apparatus cis face. These vesicles are named transition vesicles and sometimes they may fuse to form a network named transition reticulum. The third element of the Golgi apparatus is represented by bigger vesicles which separate from the trans face. They also may form a network. The composition and the morphology are different for the same sack and for the different sacks as well. The peripheral parts of sacks are dilated while the central part is narrower. There is a cis-trans polarity and a maturation process during which the materials brought by the transition vesicles undergo different transformations.

11.2 Functions of Golgi apparatus

I. Functions related to cellular secretion. Golgi apparatus is the main station on the cellular secretion pathway. At its level the materials received from ER are processed, concentrated, sorted, packed and delivered to plasmalemma. 1. A. Biochemical processing of secretion proteins. Proteolysis. Proteins receive their final size by the action of specific proteases (that are different as compared to those in RER). This way, the pro-proteins formed in RER are turned in the final proteins. B. Terminal glycosylation through which are removed certain sugar residues attached on the proteins in RER: all glucose residues and part of mannose residues. Then, the
7

glycosyl-transferases transfer on these proteins terminal sugar groups transported by nucleotides like UDP and GMP. C. Glycosylation of lipids by which the glycolipids are formed (cerebrosides and gangliosides). D. Sulphation is realized exclusively in the Golgi apparatus, by sulfate-transferases which transfer sulfate groups on proteins, lipids and glycolipids; from cerebrosides sulfatides are formed. The glycosylation of lipids and the sulphation of proteins and lipids are exclusive functions of the Golgi apparatus. 2. Sorting of secretion products consists in their separation from lysosomal enzymes. 3. Concentration of secretion products takes place during their passage through the Golgi apparatus and in the secretion vesicles. This process is achieved by interactions between the secretion proteins with positive charges and proteoglycans with negative charges. The formation of these aggregates decrease the osmotic pressure and as consequence water is removed. 4. Packing in secretion vesicles in the peripheral regions of the trans Golgi sacks. 5. Sending the secretion products to plasma membrane. II. Byogenesis of lysosomes. III. Biogenesis of membranes. Intrinsic proteins of membranes arrive in transition vesicles and then in the Golgi sacks and are modified through maturation and terminal glycosylation. They are next included in vesicles that fuse with plasmalemma. Most of the membrane lipids follow the same route, but some of them are exclusively produced in the Golgi apparatus. Both the membrane lipids and proteins participate to formation of new membranes. IV. Coordination of membranes traffic within the cells (membrane trafficking). There are transition vesicles that come from ER to the cis Golgi sacks, vesicles that transport materials from one compartment to another in the Golgi complex, secretion vesicles sent to plasmalemma. On another hand, there also are vesicles that come back from the Golgi apparatus to the ER, and from plasma membrane to the Golgi apparatus. The latter vesicles ensure the membrane recycling an important process described by G.E. Palade. By this process some parts of plasmalemma are reintroduced back in the cytoplasm together with materials included from outside the cell. This process is important because it prevents the continuously increase of the plasma membrane surface. It is also important from a qualitative point of view, because the membrane components are reused.
8