Structure and functions of endoplasmic reticulum

The presentation consists of 57 slides,describes following heads
• DISCOVERY
• INTRODUCTION
• BIOGENESIS OF ER
• ISOLATION OF MICROSOMES FROM E R
• STRUCTURE
• COMPONENTS OF ER
CISTERNAE
VESICLES
TUBULES
• MAIN FUNCTION OF ER
• TYPES OF ENDOPLASMIC RETICULUM
• SMOOTH ENDOPLASMIC RETICULUM (SER)
• FUNCTIONS OF SER
• ROUGH ENDOPLASMIC RETICULUM (RER)
• FUNCTIONS OF RER
• SUMMARY
• REFERENCES
• QUESTIONS

1. STRUCTURE AND FUNCTIONS OF ENDOPLASMIC RETICULUM (E R) By Prof (Dr.) Ichha Purak Department of Botany Ranchi Women’s College,Ranchi 1/12/2020 Endoplasmic Reticulum 1

2. 1/12/2020 Endoplasmic Reticulum 2 DISCOVERY INTRODUCTION BIOGENESIS OF ER ISOLATION OF MICROSOMES FROM E R STRUCTURE COMPONENTS OF ER CISTERNAE VESICLES TUBULES Main Function of ER TYPES OF ENDOPLASMIC RETICULUM SMOOTH ENDOPLASMIC RETICULUM (SER) FUNCTIONS OF SER ROUGH ENDOPLASMIC RETICULUM (RER) FUNCTIONS OF RER SUMMARY REFERENCES QUESTION S CONTENTS

3. DISCOVERY Discovered in 1902 by Italian Scientist Emilio Verrati . Porter et. al. (1945) showed through electron micrograph that cells contain a lace like network of strands throughout the cytoplasm. Palade and Porter (1954) mentioned key characteristics of Endoplasmic Reticulum and ribosomes. In 1966 George Palade generated a tramission electron micrograph illustrating that endoplasmic reticulum is studded with granules (ribosomes ). George Palade (1974) further described structure of endoplasmic reticulum and Golgi complex. Emilio Verrati KEITH ROBERTS PORTER 1/12/2020 Endoplasmic Reticulum 3 George Emil Palade

4. 1/12/2020 Endoplasmic Reticulum 4 All eukaryotic cells have an endoplasmic reticulum (ER) except red blood cells of mammals. In animal cells ER membrane typically constitute more than half of the total membranous system of the cell . The endoplasmic reticulum (ER) is absent in the prokaryotic cell. The entire endoplasmic reticulum is enclosed by a continuous membrane and is the largest organelle of most eukaryotic cells. Its membrane may account for about half of all cell membranes, and the space enclosed by the ER (the lumen or cisternal space) may represent about 10% of the total cell volume. INTRODUCTION . The endoplasmic reticulum (ER) is an extensive system of a network of membrane enclosed branching tubules and flattened sacs (cisternae) that extends from the nuclear membrane throughout the cytoplasm .

5. 1/12/2020 Endoplasmic Reticulum 5 BIOGENESIS OF ENDOPLASMIC RETICULUM According to current concepts, membrane biogenesis is a multi-step process that involves the synthesis of a basic membrane lipids and intrinsic proteins. Then the addition of the other components, such as enzymes, sugars or lipids occur in a sequential manner. The insertion of protein into ER membrane is independent of that of the lipids. Some proteins of the ER are formed by the ribosomes in the cytosol which then become inserted into the membrane.

6. 1/12/2020 Endoplasmic Reticulum 6 ISOLATION OF MICROSOMES FROM E R Figure : 1 Rough and Smooth Regions of ER Can Be Separated by Centrifugation (From Molecular Biology of cell , 4th Edition) A)When sedimented to equilibrium through a gradient of sucrose, the two types of microsomes separate from each other on the basis of their different densities . B ) A thin section electron micrograph of the purified rough ER fraction shows an abundance of ribosome-studded vesicles. (B, courtesy of George Palade.)

7. 1/12/2020 Endoplasmic Reticulum 7 A major contribution to the study of the E R came with a protocol published by Palade outlining a method to isolate E R derived microsomes . Isolation of Rough and smooth Microsomes is done by Density Gradient Centrifugation When cells are disrupted by homogenization, the ER breaks into fragments and give many small closed vesicles (100-200 nm in diameter) called microsomes , which can easily be purified. Microsomes derived from rough ER are studded with ribosomes and are called rough microsomes . The ribosomes are always found on the outside surface, so the interior of the microsome is biochemically equivalent to the lumenal space of the ER . As rough microsomes can be readily purified in functional form ,they are useful for studying many processes performed by the rough ER Continued :

8. 1/12/2020 Endoplasmic Reticulum 8 STRUCTURE OF ENDOPLASMIC RETICULUM Endoplasmic Reticulum extends from nuclear membrane at one end and joins plasma membrane at other end by ER-plasma membrane junction( Okeke et al, 2016) , it never opens into cytoplasm. It also continues with other organelles as Golgi apparatus and mitochondria. All these structures (tubules and sacs) are held in their place by the cytoskeleton . The ER remains bound by a phospholipid membrane. Inside the membrane, a fluid-filled space exists, which is known as cisternal space or lumen. The lumen is continuous with the perinuclear space. The membranes of ER contain many enzymes, triglycerides, phospholipids and cholesterol. Most important ER enzymes include stearases , NADH-cytochrome reductase , glucose-6- phosphatase, glycosyl transferase and Mg++ activated ATPase. Unit membranes of all the cavities of Endoplasmic Reticulum are inter connected. In Electron micrograph, the cross section of endoplasmic reticulum appears as two parallel membranes separated by a narrow light space about 4 nm wide ( called as cisternae) .

9. 1/12/2020 Endoplasmic Reticulum 9 Figure 2: Relationship between the cisternal space and the cell sap Cisternal space, perinuclear space and the external environment are closely related SER-Smooth Endoplasmic Reticulum RER-Rough Endoplasmic Reticulum Figure 3: Three dimensional structure of Rough Endoplasmic Reticulum (RER) showing stacks of flattened cisternae . Ribosomes are bound on cytosolic surface of membrane .

10. 1/12/2020 Endoplasmic Reticulum 10 COMPONENTS OF ENDOPLASMIC RETICULUM On the basis of morphology, endoplasmic reticulum is made up of 3 types of structures a)Cisternae- These are long, flattened, sac like unbranched tubules, arranged parallel forming bundles, each tubule is 40-50 µm thick b) Vesicles-These are usually rounded or ovoid structures, 25-500 mµ thick in diameter, surrounded by unit membrane, often occur isolated in cytoplasm c) Tubules-These are of diverse shape and are irregularly branched, 50-100 mµ in diameter. These are found in cells that are active in synthesis of glycerids . These are also found in big pigmented epithelial cells of retina, involved in metabolism of Vitamin –A (Retinol) All the 3 components of ER are bounded by 50-60 A⁰ thick membrane. The membrane consists of 3 layers as plasma membrane, middle thin and transparent layer of phospholipids and outer and inner dense layer of proteins. d ) Perinuclear space is the space present between E R and Nucleus. There is close relationship between perinuclear space, cisternae and cell sap

11. 1/12/2020 Endoplasmic Reticulum 11 Figure -4 Components of Endoplasmic Reticulum Main Function of Endoplasmic Reticulum Synthesis and Translocation of secretory proteins across the R ER membrane. Integration of proteins into the plasma membrane. Folding and modification of proteins in the RER lumen. Synthesis of phospholipids and steroids on the cytosolic side of the SER membrane. Storage of calcium ion in the SER lumen and their regulated release into the cytosol. Carbohydrate metabolism Detoxification of drugs

12. 1/12/2020 Endoplasmic Reticulum 12 It is basically of two functionally distinct types : Smooth Endoplasmic Reticulum (SER) and Rough Endoplasmic Reticulum (RER) on absence or presence of ribonucleoprotein granules (Ribosomes) Agranular or Smooth Endoplasmic Reticulum (SER) that does not have Ribosomes attached to its surface Granular or Rough ER (RER) The endoplasmic reticulum bears ribosomes on its surface . SER and RER change into each other as per the need of the cell. TYPES OF ENDOPLASMIC RETICULUM Figure: 5 Smooth and Rough Endoplasmic Reticulum

13. 1/12/2020 Endoplasmic Reticulum 13 TYPES OF ENDOPLASMIC RETICULUM Figure 6:The endoplasmic reticulum (ER) Electron micrograph of rough ER in rat liver cells. Ribosomes are attached to the cytosolic face of the ER membrane . ( B) Electron micrograph of smooth ER in Leydig cells of the testis, which are active in steroid hormone synthesis. ( A, Richard Rodewald , University of Virginia/Biological Photo Service; B, Dan Fawcett/Photo Researchers, Inc.) Copyright © 2000, Geoffrey M Co oper.

14. 1/12/2020 Endoplasmic Reticulum 14 SMOOTH ENDOPLASMIC RETICULUM (SER) It is highly curved and tubular SER occurs mainly in tubular form and does not have ribosomes . The tubules measure 500-1000A⁰ long, forming irregular lattices. Commonly present in cells connected with steroid and lipid biosynthesis and carbohydrate metabolism . Different cells may contain different amount of SER, depending on nature and function. The smooth ER is involved in the synthesis of lipids, including cholesterol and phospholipids, which are used in the production of new cellular membrane . In certain cell types, smooth ER plays an important role in the synthesis of steroid hormones from cholesterol . In cells of the liver, it contributes to the detoxification of drugs and harmful chemicals. The sarcoplasmic reticulum is a specialized type of smooth ER that regulates the calcium ion concentration in the cytoplasm of striated muscle cells and regulate contraction of muscles.

15. 1/12/2020 Endoplasmic Reticulum 15 FUNCTIONS OF SMOOTH ENDOPLASMIC RETICULUM The Smooth Endoplasmic Reticulum plays a crucial role in various life sustaining processes, like protein transportation, steroid and carbohydrate metabolism ,drug detoxification and synthesis of phospholipids and cholesterol, which are major components of the plasma and internal membranes . Transport of proteins SER provides route of transport of metabolites as proteins within the cell through formation of vesicles that transfer proteins to various locations. SER also transports the proteins formed in Rough ER to Golgi apparatus and other parts of the cell. Steroid Metabolism SER plays a role in steroid metabolism. Many enzymes present in membrane of SER have a key role in the synthesis of cholesterol, the precursor of steroid hormones and bile acids

16. 1/12/2020 Endoplasmic Reticulum 16 Release, uptake and storage of calcium ions SER attaches receptors to calcium binding proteins of cell membrane and stores calcium in cytoplasm of striated muscle cells which helps in muscle contraction and relaxation . In muscle cells specialized ER, called sarcoplasmic reticulum (SR) sequesters Ca 2+ from the cytosol using Ca 2+-ATPase pumps. It helps in rapid responses to extracellular signals. Lipid metabolism Synthesis of fatty acids, glycerids and steroids such as cholesterol takes place in SER and special cell organelles sphaerosomes ( present in cells of endosperm, cotyledons of seeds ) Phospholipid biosynthesis is largely confined to the membranes of SER in collaboration with Golgi complex. In the liver, the SER is abundant in hepatocytes and is involved in the production of lipoproteins.

17. 1/12/2020 Endoplasmic Reticulum 17 Carbohydrate Metabolism Glycogen , the reserve food of many cells, is converted first to Glucose-1-Phosphate and then to Glucose-6-Phosphate by gluconeogenesis. The Glucose-6-Phosphate is then brought to the smooth endoplasmic reticulum (SER) where it is broken down to Glucose and Phosphate by the enzyme Glucose-6-Phosphatase present in the SER of animal cell. The Glucose thus formed is then transported first into the cisternal space and finally to the extracellular space or to the blood stream for the maintenance of the functions of red blood cells and nerve tissues There are other enzymes of carbohydrate metabolism, such as Amylase and glucuronidase for the hydrolysis of starch and mucopolysaccharides

18. 1/12/2020 Endoplasmic Reticulum 18 Detoxification of Toxic Chemicals It detoxifies the toxicity of carcinogens by having necessary enzymes. The toxic chemicals are secreted out of the body through formation of vesicles by SER SER helps in detoxification of a wide variety of x enobiotics such as barbiturates, ethanol, aspirin and petroleum products. Detoxification generally takes place in liver cells . When large quantities of toxic harmful compounds enter the circulation, detoxification enzymes are synthesized in large quantities and smooth ER doubles its surface area within a few days. Once these harmful compound are detoxified and removed, the excess smooth ER is removed by lysosome dependent autophagocytosis . Detoxification is carried out by a system of oxygen transferring enzymes ( oxygenases ) that catalyze a number of reactions that can make lipid soluble drugs and metabolic wastes into water soluble, so that they can be easily expelled from the body.

19. 1/12/2020 Endoplasmic Reticulum 19 The enzymes present in SER help in converting lipids and other hydrophobic compounds to water soluble form for easy transport through the membrane. These oxidase enzyme systems for the detoxification of drugs and other chemicals, consist of an important element, termed cytochrome P 450. This cytochrome P450 has an important role in the detoxification of drugs. Cytochrome P450 is an iron containing compound having a special property of reducing its form when it binds with the substrate. This reduced form again can absorb light at 450 nm. When any compound, say drug, is attached with the cytochrome P450, the iron (Ferric, Fe+++) present is reduced to Ferrous (Fe++) with the help of NADPH dependent cytochrome P450 reductase . Now the reduced cytochrome P 450 will bind oxygen. One atom of oxygen is used to oxidize the substrate to convert the (Fe++) to (Fe+++) and the other atom of oxygen is used to oxidize the drug with the liberation of one molecule of water.

20. 1/12/2020 Endoplasmic Reticulum 20 Other functions of SER It helps in exchange of materials between nucleoplasm and cytoplasm. In plant cell SER establishes link between cytoplasm of two cells through pits and plasmodesmata . Smooth Endoplasmic Reticulum (SER) is responsible for the synthesis and repair of membranes. SER and RER provide surface area for the action and storage of enzymes and their products. In addition to cytochrome P 450, cytochrome P 448 and cytochrome b5 are also present in the endoplasmic reticulum. Cytochrome b5 has been employed for the desaturation of fatty acids. Cytochrome P448 tries to metabolize polycyclic hydrocarbons.

21. 1/12/2020 Endoplasmic Reticulum 21 ROUGH ENDOPLASMIC RETICULUM RER (Rough Endoplasmic Reticulum) is characterized by presence of ribosomes over the surface, connected with production of secretory proteins and is very common in cells involved in growth and differentiation. Rough ER is named for its rough appearance, which is due to the ribosomes attached to its outer (cytoplasmic) surface. Rough ER lies immediately adjacent to the cell nucleus, and its membrane is continuous with the outer membrane of the nuclear envelope. RER is found throughout the cell but the density is higher near the nucleus and the golgi apparatus. Ribosomes are ribonucleoprotein particles present either free in cytoplasm or attached to Endoplasmic Reticulum or outer surface of nuclear membrane. Ribosomes are place for protein synthesis and synthesize structural proteins when present free in cytoplasm and secretory proteins when attached to E R .

22. 1/12/2020 Endoplasmic Reticulum 22 FUNCTIONS OF ROUGH ENDOPLASMIC RETICULUM Protein synthesis, transport and folding Proteins that are secreted from the cells, or act as integral membrane proteins or proteins of certain organelles like Golgi complex, lysosomes, endosomes are assembled on ribosomes attached to the outer surface of RER membranes. RER is more prominent in liver cells, as they are concerned with the production of proteins. So, cells specializing in protein synthesis have more prominent RER, while SER is prominent in those cells, which are concerned with the production of lipids . Thus, these proteins, instead of passing into the cytoplasm, appear to pass into the cisternae of the RER, and are protected from action of cytoplasmic protease enzyme.

23. 1/12/2020 Endoplasmic Reticulum 23 Signal sequence vary in length (13-36AAs, having about 10-15 hydrophobic AAs, one or two + ve AAs before hydrophobic sequence, a short sequence of polar AAs near C terminus and Amino Acid with short side chain (Alanine) closest to cleavage site. These proteins contain a signal sequence of 6 to 20 non-polar amino acid residues. It targets the nascent polypeptides for the ER membrane and leads growing polypeptide into the ER lumen. SIGNAL SEQUENCE Figure : 7 Structure of Signal Sequence

24. 1/12/2020 Endoplasmic Reticulum 24 SRP is a rod shaped complex having single 7S RNA ( 300 nucleotides) and 6 different proteins. One protein of SRP directly binds to signal sequence, inhibiting elongation by strictly blocking entry of Amino acyl tRNAs inhibiting peptidyl transferase Figure: 8 SIGNAL RECOGNITION PARTICLE (SRP) A signal recognition particle (SRP), recognizes the signal sequence and acts as a tag that allows the SRP-ribosome-nascent polypeptide to bind to an SRP receptor located on the cytosolic surface of the ER membrane

25. 1/12/2020 Endoplasmic Reticulum 25 As one nascent polypeptide enters the RER cisternae, it acts upon by a variety of enzymes located either within the membrane or in the lumen of the RER. These enzymes transform the nascent proteins into their proper functional state . Integral membrane proteins, such as glycophorin and a fraction of the erythrocyte plasma membrane are synthesized on membrane bound ribosomes and translocated through the ER membrane as the polypeptides elongate. Integral proteins contain one or more stretches of hydrophobic amino acids that serve as stop transfer sequences, which block further movement of these proteins into the cisternal chamber, but trigger the opening of the channel laterally and cause the inser­tion of the hydrophobic helix into the lipid bilayer

26. 1/12/2020 Endoplasmic Reticulum 26 RER plays a central role in the synthesis and export of proteins and glycoproteins in secretory cells as for example liver cells secreting serum proteins (albumin), endocrine cells secreting peptide hormones (insulin), pancreatic acinar cells secreting digestive enzymes, and cartilage cells secreting collagen. These proteins are glycoproteins, membrane proteins, lysosomal proteins, albumins, globulins etc. Ribosome become attached to E R when secretory protein is needed by the cell , for this ribosomes receives specific signals. The ribosomes on rough ER specialize in the synthesis of proteins that possess a signal sequence that directs them specifically to the ER for processing. (A number of other proteins in a cell, including those destined for the nucleus and mitochondria, are targeted for synthesis on free ribosomes, or those not attached to the ER membrane).

27. 1/12/2020 Endoplasmic Reticulum 27 Figure11 : Overview of Protein sorting From Chapter 9 : Protein sorting and Transport from the book : Cell -A Molecular Approach by G M Cooper Page 149

28. 1/12/2020 Endoplasmic Reticulum 28 The role of ER in protein processing and sorting was first demonstrated by George Palade and his colleagues in the 1960s . He defined the pathway for secretory proteins on the basis of some experiments Rough ER → G A →Secretory vesicle → Exterior The entrance of proteins into the ER thus represents a major branch point for the traffic of proteins within eukaryotic cells, which correspond to the synthesis of proteins on either free or membrane bound ribosomes. Proteins synthesized by the rough ER have specific final destinations. Some proteins, for example, remain within the ER, whereas others are sent to the Golgi apparatus, which lies next to the ER. Proteins secreted from the Golgi apparatus are directed to lysosomes or to the cell membrane; still others are destined for secretion to the cell exterior.

29. 1/12/2020 Endoplasmic Reticulum 29 Gunter Blobel David D Sabatini Gunter Blobel and David D Sabatini ( 1971 ) proposed signal hypothesis. The mRNA translating secretory proteins on E R membrane bound ribosomes contain a unique set of codons ( signal codons ) at the 3’ end just after initiation codon which produces signal sequence at N(Amino) terminus of Growing polypeptide chain which is recognized by SRP and Places ribosome on ER membrane for completing polypeptide synthesis in lumen of ER .

30. 1/12/2020 Endoplasmic Reticulum 30 Targeting Proteins to the Endoplasmic Reticulum All protein synthesis initiates on ribosome that are free in the cytosol. Ribosomes engaged in the synthesis of proteins that are destined for secretion are then targeted to endoplasmic reticulum due to presence of a Signal Sequence at the amino terminus of the growing polypeptide chain . This signal sequence is a short stretch of hydrophobic amino acids. It is translated due to presence of signal codons on some specified messenger RNAs The Signal hypothesis was proposed by Gunter Blobel and David D Sabatini in 1971 as mRNAs to be translated on membrane bound ribosomes contain a unique set of codons at the 3’ end just after initiation codon Translation of these codons yield a unique sequence at the amino terminus of the growing polypeptide chain (the signal sequence ) . This signal sequence triggers attachment of ribosome to the membrane of ER with the help of SRP ( Signal Receptor Protein ) . The signal sequence is cleaved later on by signal peptidase after the targeting function is completed

31. 1/12/2020 Endoplasmic Reticulum 31 Figure: 10 Schematic diagram showing the mechanism by which signal sequence directs the destiny of protein through lumen of Endoplasmic Reticulum

32. 1/12/2020 Endoplasmic Reticulum 32 Figure: 11 Targeting secretary proteins to the Endoplasmic Reticulum(E R ) From Chapter 9 : Protein sorting and Transport from the book : Cell -A Molecular Approach by G M Cooper Page 153 Steps 1 . As the signal sequence emerges from the ribosome, it is recognized and bound by Signal Recognition Particle (SRP ) 2. The SRP escorts the complex to the ER membrane, where it binds SRP receptor 3. The SRP is released, the ribosome binds to membrane translocation complex and the signal sequence is inserted into membrane channel. 4. Translation resumes and the growing polypeptide chain is translocated across the membrane 5. Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of the ER

33. 1/12/2020 Endoplasmic Reticulum 33 steps in synthesis of secretory proteins Initiation complex formation (mRNA-ribosome ,I tRNA ) Protein synthesis initiates If signal sequence appears at amino terminus of nascent polypeptide SRP binds to signal sequence of growing polypeptide Ribosome stops further elongation of polypeptide (Translation is blocked) for a while SRP is recognized by a Docking protein present on E R membrane Ribosome is attached to E R membrane and rest on ribosome receptors SRP dissociates and is recycled protein synthesis is resumed Translocation of polypeptide into the lumen of ER takes place through a channel (pore) created Signal sequence is cleaved by enzyme signal peptidase present in lumen of E R Polypeptide is cleaved , ribosome dissociates Polypeptide chain after being released into the lumen of ER is further modified , chain folds due disulphide and other bonds. Many proteins are also glycosylated .

34. 1/12/2020 Endoplasmic Reticulum 34 Synthesis of Membrane Lipids The enzymes involved in the synthesis of phospholipids are themselves integral proteins of the RER membrane with their active sites facing the cytosol. . Vesicular Transport The cisternae of the RER are typically large with flattened cavities that can act as transport channels for membrane and luminal proteins to be moved from their site of synthesis to the apical tips of the RER. The apical edges of the RER cisternae are typically smooth, devoid of ribosomes and are referred to as transitional elements, which serve as sites of formation of the first set of transport vesicles in the biosynthetic pathway leading towards Golgi complex. Formation of Tertiary Structure of Protein ( Protein folding) If a polypepetide is translocated into the ER from a membrane bound ribosome it is known as co-translational translocation. If a polypeptide is incorporated into the ER from a free ribosome it is known as post- tranlational translocation Protein folding involves disulphide bond formation by the enzyme disulphide isomerase of E R between two cysteine molecules present distantly on polypeptide chain .

35. 1/12/2020 Endoplasmic Reticulum 35 . This requires an oxidising environment of ER , whereas cytosol provides a reducing environment The folding of a polypeptide into its correct three dimensional protein structure is mediated by molecular chaperones. Chaperones bind to unfloded polypeptides, stabilising them to prevent incorrect folding. One of the primary chaperone proteins is Binding immunoglobulin Protein ( BiP ). BiP belongs to a heat sensitive protein family (hsp70) and has both a protein binding and an ATPase domain. BiP is also used to seal the translocon pore when not occupied by a ribosome.

36. 1/12/2020 Endoplasmic Reticulum 36 Nearly all the proteins produced on the membrane bound ribosomes are glycoproteins. The addition of sugars is catalyzed by a group of membrane bound enzymes called glycosyl transferase in ER or Golgi apparatus. Glycoproteins often are linked to oligosaccharides through Asn ( Aspargin ) residues. through the calnexin / calreticulin cycle In almost all glycoproteins a 14 residue core (tree) oligosaccharide consisting of 2 acetyl glucoseamine , 9 mannose, 3 glucose is added to protein. The presence of one or more mannose 6P residue in their linked oligosaccharide is the structural signal that targets these proteins into lysosome There are two types of protein glycosylation for polypeptides imported into ER lumen . First type is N-linked glycosylation which actually begins in the ER by a co-translational mechanism whereas second type is O-linked glycosylation occurs only when after polypeptide is transported into Golgi apparatus by a post translational mechanism N-linked Protein Glycosylation Begins in the ER

37. 1/12/2020 Endoplasmic Reticulum 37 Figure: 12 B Proteins move from the ER to the golgi apparatus and then , within secretory vesicles to the plasma membrane and cell exterior Figure: 12A Interrelated position of golgi complex, endoplasmic reticulum and nucleus in an animal cell A receptor protein in the membrane of Golgi complex recognizes this mannose 6P signal. Proteins are moved from ER to Golgi Complex in transport vehicles. Glycosylation plays a key role in protein targeting

38. 1/12/2020 Endoplasmic Reticulum 38 The proximity of the rough ER to the cell nucleus gives the ER unique control over protein processing . The rough ER is able to rapidly send signals to the nucleus when problems in protein synthesis and folding occur and thereby influences the overall rate of protein translation . When misfolded or unfolded proteins accumulate in the ER lumen, a signaling mechanism known as the unfolded protein response (UPR) is activated. The response is adaptive, such that UPR activation triggers reductions in protein synthesis and enhancements in ER protein-folding capacity and ER-associated protein degradation . If the adaptive response fails, cells are directed to undergo apoptosis (programmed cell death). Unfolded Protein Response (UPR )

39. 1/12/2020 Endoplasmic Reticulum 39 The ubiquitin ( Ub )-proteasome pathway (UPP) of misfolded proteins degradation . Ubiquitin is conjugated to misfolded proteins that are destined for degradation by an ATP-dependent process that involves three enzymes . A chain of five Ub (ubiquitin) molecules provides a recognition signal for the 26S proteasome ,where receptors initiate degradation of proteins into smaller peptides and finally into amino acids by peptidases/proteases

40. RIBOSOME AND POLYSOMES /POLYRIBOSOMES Ribosomes are Ribonucleoprotein non membranous particles . Ribosomes are the site of Protein Synthesis Ribosomes are present in cytoplasm either free or attached to ER or nuclear membrane. In eukaryotic cells ribosomes are also present in mitochondria and plastids. Prokaryotic ribosomes are 70S particles having 2 subunits 30 S & 50S Eukaryotic cytoplasmic ribosomes are 80S particles having 2 subunits 40S & 60 S The two sub units are dissociated at low Mg++ concentrations and associate at high Mg++ concentrations. The smaller subunit fits on larger subunit leaving a tunnel between two,which can accommodate the mRNA , aminoacyl tRNAs and other factors during protein synthesis . 40 Appendix 1/12/2020

41. Both the sub unit of ribosome contain rRNAs and proteins Ribosomes are dumb bell shaped or spherical having a diameter of 140A-160A ᵒ Number of ribosomes/cell is directly related to RNA content and protein synthesis rates. Ribosomal RNA are large molecules ,larger than diameter of ribosome,remains coiled like a spring with helical and non helical regions. rRNA of smaller unit helps in correct orientation of mRNA on ribosome during protein synthesis 2 ribosomal units after organisation in nucleolous pass to cytosol and aggregate to form complete functional ribosomal complex in cytosol . Many ribosomes can bind same messenger RNA one after one for polypeptide synthesis of same nature forming polysome 41 Appendix 1/12/2020

42. 42 Appendix 1/12/2020 Figure: 13 proteins and RNAs of Prokaryotic and Eukaryotic ribosomes

43. 1/12/2020 Endoplasmic Reticulum 43 Both in case of free and membrane bound ribosomes, when the ribosomes moves some distance on mRNA, a second ribosome may bind with the help of initiation codons, so many ribosomes may use same mRNA for polypeptide synthesis of same nature differing only in length of PPC (Number of amino acids polymerized). This assemblage is referred as polysome or polyribosome POLYRIBOSOME Figure : 14 Formation of Polyribosomes

44. 1/12/2020 Endoplasmic Reticulum 44 All of cell’s lipids are produced in ER . Synthess of a major portion of cell’s proteins occurs on the cytosolic surface of the ER . These proteins are destined for secretion through ER to Golgi apparatus , Lysosomes ,endosomes and plasma membrane. In the ER lumen, the proteins fold and oligomerize , disulfide bonds are formed, and N-linked oligosaccharides are added. N-linked glycosylation is used to indicate the extent of protein folding, so that proteins leave the ER only when they are properly folded. Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are deglycosylated , ubiquitylated , and degraded in proteasomes . Only proteins that carry a special ER signal sequence are imported into the ER. The signal sequence is recognized by a signal recognition particle (SRP), which binds both the growing polypeptide chain and ribosome and directs them to a receptor protein on the cytosolic surface of the rough ER membrane. SUMMARY

45. 1/12/2020 Endoplasmic Reticulum 45 This binding to the ER membrane initiates the translocation process by threading a loop of polypeptide chain across the ER membrane through the hydrophilic pore in a transmembrane protein translocator . Soluble proteins—destined for the ER lumen, for secretion, or for transfer to the lumen of other organelles—pass completely into the ER lumen . Transmembrane proteins destined for the ER or for other cell membranes are translocated partway across the ER membrane and remain anchored there by one or more membrane-spanning α-helical regions in their polypeptide chains . These hydrophobic portions of the protein can act either as start-transfer or stop-transfer signals during the translocation process. When a polypeptide contains multiple, alternating start-transfer and stop-transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein.

46. 1/12/2020 Endoplasmic Reticulum 46 Blobel G and Sabatini D D (1971). Ribosome-membrane interaction in eukaryotic cells. Biomembranes , 2, 193–195 Blobel G and Dobberstein B ( 1975). "Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma". Journal of Cell Biology. 67 (3): 835–851 Okeke et al (2016) Endoplasmic reticulum–plasma membrane junctions: structure, function and dynamics. J Physiol.594(11): 2837-2847 Palade G E and Porter KR. (1954) Studies on the endoplasmic reticulum. I. Its identification in cells in situ. J Exp Med. ;100(6):641–656 . Palade G E (1974) Intracellular aspects of the process of protein secretion –Nobel Prize lecture,published in 1992 by the Nobel Prize Foundation Porter KR, Claude A, Fullam EF (1945). "A Study of Tissue Culture Cells by Electron Microscopy". J Exp Med. 81 (3): 233–246. Porter KR ( 1953). "Observations on a submicroscopic basophilic component of cytoplasm". The Journal of Experimental Medicine. 97 (5): 727–750 . Veratti E., 1902. Ricerche sulla fine struttura della fibra muscolare striata. Mem. R. Ist. Lombardo, Cl. Sc. Mat. Nat., 19: 87-133 . REFERENCES

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48. 1/12/2020 Endoplasmic Reticulum 48 QUESTIONS ON ENDOPLASMIC RETICULUM MCQs on ER 1) Which of the following is the largest single membrane-bound intracellular compartment? a) Ribosome b) Golgi apparatus c) Nucleus d) Endoplasmic reticulum 2) Endoplasmic reticulum membrane which is associated with ribosomes is called_______ a) ER lumen b) Smooth endoplasmic reticulum c) Rough endoplasmic reticulum d) Endosome 3 ) Which of the following is not the function of Glycosylation? a) Helps in proper folding of the protein b) Confer stability in proteins c) Helps in cell-cell adhesion d) Synthesis of membrane lipid 4) Name the site where detoxification of xenobiotic compounds takes place? Cytosol b) RER c) SER d) Ribosome 5) Name the sequence which allows the resident protein to retain in ER lumen? a) KDEL b) KKXX c) KLDE d) KXXK 6) Which of the following coated vesicle transport protein from ER to Golgi? a) Clathrin b) COP II c) COP I d) COP III 7) Protein folding is a process in which a polypeptide folds in to ___________ a) 2-D structure b) Globular form c) 3-D structure d) Linear form

49. 1/12/2020 Endoplasmic Reticulum 49 8) Name the coated vesicle which is used to transfer protein from plasma membrane to endosome? a) Clathrin b) COP I c) COP II d) COP III 9) Smooth form of endoplasmic reticulum is without a) Golgi complex b) Ribosome c) Nucleus d) Mitochondria 10) Rough endoplasmic reticulum assists in the synthesis of a) Carbohydrates b) Proteins c) fats, glucose, starch d) Lipids 11) Secretions produced by ribosome are passed via endoplasmic reticulum and Golgi complex b) Mitochondria c) Nucleus d) Vacuole 12) The term Endoplasmic Reticulum was coined by a) Camillo Golgi b) Porter c) Robert Brown d) Benda 13) Which of the following organelle has a continuous connection with nuclear membrane Golgi apparatus b) Lysosome c) RER d) SER 14) In RER, ribosomes are located on a) the cytoplasmic side b) on the luminal side c) both (a) and (b) d) all throughout 15) Which of the following statements were true regarding ER a) ER provides structural framework to the cell b) ER acts as intra cellular transporting system c) SER is involved in the synthesis of lipid d) All of the above

50. 1/12/2020 Endoplasmic Reticulum 50 16 ) The rough ER is specially well developed in cells actively engaged in Protein synthesis b) Nucleotide synthesis c) Lipid synthesis d) Secretory functions 17) The term Endoplasmic Reticulum was coined by a ) Reinert b) Johnson c) Pomaret d) Porter 18) Which of the following statements are incorrect regarding Endoplasmic Reticulum ? a) The adipose tissue has both SER and RER b ) Plasma cells has RER only. RBC lacks both RER and SER d ) Hepatocytes has both RER and SER 19) SER is involved in Phospholipid biosynthesis and detoxification reaction b) Protein synthesis Phospholipid biosynthesis and Protein synthesis d ) Phospholipid biosynthesis 20) The functions of RER include a)Protein synthesis and detoxification b)Protein synthesis and post translational modification c) Protein synthesis and phospholipid biosynthesis d) Protein synthesis only 21) Which of the following organelle is involved in xenobiotic detoxification a) Golgi body b ) Lysosome c ) SER d ) RER 22) Protein glycosylation occurs in a)Lumen of mitochondria b) Lumen of RER c)Lumen of SER d) Lumen of Lysosome

51. 1/12/2020 Endoplasmic Reticulum 51 23) Ribophorins are a) Transmembrane glycophorin on RER b) Transmembrane Glycophorin on SER Luminal proteins on RER d) Luminal proteins on SER 24) RER is involved in the synthesis of a) Membrane proteins and Secretory proteins b) Different proteins of the cell c) Membrane proteins, secretory proteins and lysosomal proteins Membrane proteins and secretory proteins and nuclear proteins 25) Rough ER is called ‘rough’ because a ) Rough texture of the surface b) Surface is studded with membrane proteins c ) Surface is studded with ribosomes d ) All of these 26) SER produces a)Proteins b)Carbohydrate c) Lipid d ) Nucleic acid 27) The main organelle involved in modification and routing of newly synthesized proteins to their destination is a) Chloroplast b) Mitochondria c) Lysosome d) Endoplasmic Reticulum 28) The transfer vesicle from RER fuse with which region of golgi complex a) Cis b) Medial c) Trans d) Protein arms

52. 1/12/2020 Endoplasmic Reticulum 52 29) Which of the following is related to glycosylation of proteins a) Endoplasmic Reticulum b) Peroxisome c) Lysosome d) Mitochondria 30) Sarcoplasmic reticulum is related with a)Protein synthesis b) Hormone synthesis c) Release of Ca ++ ions and contraction of muscles d) None of these 31) During ultracentrifugation the Endoplasmic Reticulum and bodies associated with it are separated as a fraction known as a) Microsomes b) Polysome c) Quantosome d) Episome 32) The most important function of ER is a) Protein synthesis b) Nourishing the nucleus c) Secretion of materials d) To give shape to the cell 33) In rapidly dividing cells, ER is a) Highly developed b) Poorly developed c) Absent d) Non-functional 34) In endoplasmic Reticulum the following process takes place a)Lipid synthesis b) Channeling of biosynthetic process c) Steroid synthesis d) All of the above 35) Single unit membrane structure present in the cytoplasm in the form of a net is a)Golgi Complex b) Peroxisome c) Lysosomes d) Endoplasmic Reticulum

53. 1/12/2020 Endoplasmic Reticulum 53 36) The Endoplasmic Reticulum often bears Lysosomes b) Centrioles c) Peroxisomes d) Ribosomes 37) When the region of endoplasmic reticulum are studded by ribosome on the outer surface of the cisternae, it is called a)Sarcoplasmic Reticulum b) SER c) Granular Endoplasmic Reticulum d) None of the above 38) Which of the following is responsible for mechanical support, enzyme transport and protein synthesis a) Dictyosome b) Cell membrane c) Mitochondria d) Endoplasmic reticulum 39) Less stable Endoplasmic Reticulum is a ) RER b ) SER c ) Cisternae d ) Tubules 40) Endoplasmic Reticulum is in continuation with Golgi body b) nuclear wall c)Mitochondria d ) Cell wall 41) A eukaryotic cell without endoplasmic reticulum is a) Egg cell b) RBC of mammals c) WBC d) None of the above 42) Ribosomes are attached to endoplasmic reticulum through a) Ribophorins b) r-RNA c) t -RNA d) hydrostatic reactions

54. 1/12/2020 Endoplasmic Reticulum 54 43) Polyribosomes are aggregation of Ribosome and r-RNA b ) only r-RNA c) peroxisomes d ) Several ribosomes held together by a string of m-RNA 44) Which of the following organelles has the function of storing intracellular calcium? a) Lysosome b) Endoplasmic reticulum c) Nucleolus d) Golgi complex 45) Which component of the Golgi apparatus is primarily responsible for "receiving" proteins to be packaged and modified? a) Cis Golgi b) Medial Golgi c) Trans Golgi d) Vacuoles 46) Following are the membrane bound cell organelles except Endoplasmic reticulum b)Lysosome c) Ribosomes d) Peroxisome 47) ---------------- is the organelle which produces transition vesicles to transport molecules produced in ER to the Golgi. a) Lysosome b) Mitochondria c) Smooth ER d) Nucleus 48) 1) Which is the site of protein synthesis in a cell? a ) Lysosome b) Golgi body c) Smooth endoplasmic reticulum d)Ribosomes 49) ---------------- is the organelle responsible for the origin of other membranes in the cell. a ) Mitochondria b) Smooth endoplasmic reticulum c)Golgi body d) Lysosome 50) ) -------------- is an important element of oxidase enzyme present in the ER which is involved in the modification of drugs. a ) Cytochrome P450 b) CYP3A4 c) Aromatase d)M21- Hydroxylase Answers : Note : Options in red colour are correct choice for MCQs

55. 1/12/2020 Endoplasmic Reticulum 55 Short Answer Questions Write short notes on the following a) Granular Endoplasmic reticulum b) Agranular Endoplasmic Reticulum c) Role of RER in synthesis of secretory proteins d) Structure of Signal Recognition Particle (SRP) e) Receptor Protein on membrane of E R f) Signal Hypothesis g) Structure of ribosome h) Polyribosome i) Diagram of Endoplasmic Reticulum Components of E R Explain the following in brief a) Role of SER in detoxification b) Signal sequence c) Structure of Signal Recognition Particle (SPR) d) Role of Signal Recognition Particle in protein targeting e) Functions of SER f ) Structure of Signal peptide

56. 1/12/2020 Endoplasmic Reticulum 56 Long Answer Questions 1)Give an account of ultrastructure of endoplasmic reticulum and describe its function. 2) What do you mean by membrane system ? Describe the same with reference to Endoplasmic Reticulum. Describe the structure and function of Endoplasmic Reticulum 4) Explain the types of Endoplasmic Reticulum and their function . 5) Discuss the functions of smooth endoplasmic reticulum . 6) Discuss the functions of Rough endoplasmic Reticulum . 7) Give the structure of endoplasmic reticulum and point out the significance of ribosomes found attached to it .

57. 1/12/2020 Endoplasmic Reticulum 57 THE END THANKS