Endoplasmic Reticulum (ER) PDF

BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM TOPIC 7: THE ENDOPLASMIC RETICULUM (ER) 1. THE ENDOMEMBRANE SYSTEM 2. THE ENDOPLASMIC RETICULUM (ER) 2.1. Rough Endoplasmic Reticulum (RER) 2.2. Smooth Endoplasmic Reticulum (SER) 3. SECRETORY AND...

BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM TOPIC 7: THE ENDOPLASMIC RETICULUM (ER) 1. THE ENDOMEMBRANE SYSTEM 2. THE ENDOPLASMIC RETICULUM (ER) 2.1. Rough Endoplasmic Reticulum (RER) 2.2. Smooth Endoplasmic Reticulum (SER) 3. SECRETORY AND CYTOSOLIC PATHWAYS FOR PROTEIN SYNTHESIS 3.1. Cytosolic pathway 3.2. Secretory pathway 4. RER: THE START OF THE SECRETORY PATHWAY 5. CYTOSOLIC vs. RER POLYRIBOSOMES 5.1. Characteristics 6. TRANSLOCATION OF PROTEINS 6.1. Synthesis of water soluble proteins 6.2. Synthesis of transmembrane proteins 6.3. Glycosylation 7. FOLDING AND QUALITY PROTEINS 7.1. Chaperons and protein folding 7.2. The unfolded protein response (UPR) 8. SMOOTH ENDOPLASMIC RETICULUM (SER) 8.1. Cytochrome p450 system 8.2. Membrane lipid biosynthesis in the SER 9. INTEREXCHANGE OF LIPIDS BETWEEN THE ER AND OTHER ORGANELLES 10. OTHER ER FUNCTIONS: STORAGE OF Ca2+ 1. THE ENDOMEMBRANE SYSTEM The endomembrane system consists of a group of organelles which are interconnected through transport vesicles and work together to modify, package and transport lipids and proteins. They are independent but work together. - These organelles are the endoplasmic Reticulum, (nuclear envelope), the golgi apparatus, endosomes, phagosomes, autophagosomes, lysosomes and transport vesicles. → these communicate with each other through vesicles. - It is extremely important to remember that peroxisomes, mitochondria and chloroplast do NOT belong to the  endomembrane system, because they don’t receive vesicles from the other organelles. BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM 2. THE ENDOPLASMIC RETICULUM (extension of the nuclear envelope) It is a continuous membrane system that forms a series of flattened sacs within the cytoplasm of eukaryotic cells and serves multiple functions, being important particularly in the synthesis, folding, modification, and transport of proteins and lipids too. It is a complex membrane organelle, and they have a very variable development in the cell depending on the cell type/activity (especially abundant in protein and lipid secreting cells). It’s presence is depending on the cell type, especially cells that have to secrete proteins or lipids will have a big ER. 2.1. RER: ROUGH ENDOPLASMIC RETICULUM - It has ribosomes attached to the membrane. - Cisterns are aligned in parallel. - Main function → involved in protein metabolism: - Protein synthesis: beginning of secretory pathway. - Protein sorting: It sorts the proteins. - Protein glycosylation: The amino acids have sugar molecules attached to them. - Protein folding: There is a high amount of chaperons. Proteins need to be properly folded so they have a specific fufunction - Synthesized proteins are stored in the lumen of the ER and will reach the Golgi apparatus and other organelles through vesicles. 2.2. SER: SMOOTH ENDOPLASMIC RETICULUM - It does not have ribosomes attached to it - Cisterns are more twisted, they look more disorganized. It is more irregular (in cross section it looks like there is an accumulation of vesicles). - Main function → lipid metabolism - Membrane lipid synthesis (when the lipids are reduced they are send to other organelles by means of vesicles or other translocators). - Interexchange of lipids between organelles ( through vesicles or specific cytosolic transporters) - Phospholipids, sphingolipids, cholesterol and Steroid hormones (cortisol…) synthesis - Detoxification of toxic organic compounds through oxidation (enzymes of the cytochrome P450) !!! RER/SER ratio depends on the cell type and its physiological function. For example, cells synthesizing large amounts of proteins will have a very developed RER and cells having a very high lipid metabolism will contain a very developed SER. There is continuity between the SER and the RER, they are arranged differently but they are communicated. 3. SECRETORY AND CYTOSOLIC PATHWAYS FOR PROTEIN SYNTHESIS  Ribosomes initiate translation in the cytosol, after that, there are two routes that proteins use to reach their destination within the cell:: BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM 3.1. CYTOSOLIC PATHWAY: Direct translation of the mRNA. The ribosome can continue being a free ribosome and the synthesis of the protein is going to finish in the cytoplasm. Proteins following this route are synthesized by free ribosomes in the cytosol. These proteins usually remain in the cytoplasm or are directed to organelles like mitochondria, nucleus or peroxisomes (none of them is an organelle of the endomembrane system), were they perform specific functions. The proteins are released to the cytosol and need to be exported to the organelles by different mechanisms. This pathway is common for proteins that do not require extensive post-translational modifications. 3.2. SECRETORY PATHWAY: The translation of mRNA starts on the surface of the ER, followed by transport through vesicles to the Golgi apparatus. Here, proteins are modified and packaged into secretory vesicles, which will fuse with the plasma membrane to release the proteins outside the cell. This pathway is typical for proteins that need to be secreted, sent to specific organelles or targeted to lysosomes. This proteins have specific signals: - GLYCOSYLATIONS. All proteins will have sugars residues attached to them (it is their main characteristic). These glycosylations will start in the ER and they will be finished in the Golgi apparatus. 4. RER: THE START OF THE SECRETORY PATHWAY Ribosomes attached to its surface (electron dense spots) will be translocated to the RER lumen (cistern inside), which will contain newly synthesized proteins along with chaperons. The RER consists of highly aligned cisterns charged, visualized in parallel and they have electrodense spots (ribosomes).  Synthesis starts in the cytosol, the ribosome and the mRNA bind in the cytosol, (the first amino acids are translated by cytosolic ribosomes), but the ribosome is immediately translocated to RER thanks to an HYDROPHOBIC SIGNAL PEPTIDE (8-10 hydrophobic amino acids) → (this will be the ER import signal) →if anything has this signal, it will be translocated to the ER. BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM 5. CYTOSOLIC vs. RER POLYRIBOSOMES Many ribosomes read one mRNA molecule at a time. In the secretory pathway, in order for the mRNA-ribosome complex to be translocated to the ER, the protein needs to have one specific signal (SRP) which bounded to the signal peptide creates the RER import signal (close to NH2 protein end). 5.1. CHARACTERISTICS - 8 to 10 hydrophobic amino acids in a row (Leu, Ile, Phe, Trp, Val, Met, Ala): hydrophobic block - It’s a consensus sequence (not fixed: relative changes are allowed as long as they are hydrophobic amino acids.) - If you remove it from a gene → block translocation (it shows that sequences are fundamental to immediate translocations). Proteins rely on this system to reach the ER. - A special protein (SRP, signal recognition particles) recognises and binds to these hydrophobic peptide sequences. So thanks to this movement of the SRP molecule, we are going to have ribosomes attached to the rough endoplasmic reticulum (RER). 6. TRANSLOCATION OF PROTEINS Shortly after the initiation of translation the signal peptide, which has to be hydrophobic, will appear. Typically, this peptide will have a consensus sequence created by 8-10 amino acids (ex: Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Tyr). This will be recognized by the Signal Recognition Particle (SRP). This protein, first of all, recognizes the signal sequence of the nascent newborn protein, it gets in touch with it, and also with the ribosome that is free in the cytosol. Finally, it is going to transport both the nascent protein with the ribosome and the mRNA molecule towards an SRP receptor, and this receptor is located in the RER. So, in other words, SRP binds the peptide and brings the mRNA-ribosome complex to the RER surface. Thanks to this movement of the SRP molecule, and to the interaction of the SRP with the protein translocation machinery in the ER, we are going to have ribosomes attached to the rough endoplasmic reticulum. Translation will be transiently stopped until the whole complex gets bound to the ER membrane. Once the ribosome together with the nascent protein is attached to the rough ER, there is a cross membrane BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM protein called translocon (which will catalyze translocation of the new protein to the ER lumen) is going to open. Once this protein opens, the synthesis of the protein is not going to happen anymore in the cytosol, but inside the lumen of the endoplasmic reticulum. → In the process, there could be different situations depending on the protein. 6.1. SYNTHESIS OF WATER SOLUBLE PROTEINS Ribosome is going to be bound to the ER. Then, the signal peptide will interact with the translocon in a way that the edge of the protein will be sticking facing the cytosolic side. Meanwhile the ribosome will resume translation so new amino acids will be added to the newborn protein and after a while, signal peptides will come and cleave the protein by the signal peptide depart. Like this, it will remove the signal peptide from the new protein (is always removed). In the process, the signal peptide will remain bound to the translocator, but the protein will be released free in the ER lumen. 6.2. SYNTHESIS OF TRANSMEMBRANE PROTEINS Transmembrane proteins are those who are permanently attached to the membrane by hydrophobic domains but cross to the lipid bilayer. Transmembrane proteins are different, because apart from the first hydrophobic block (there are several), they have other internal signal sequences: the start and stop transfer sequences. The stop signals bind to the proteins too and the part that has been synthesized stays inside and the part that is going to be done gets out. BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM Single Stop sequence: The single-pass transmembrane protein will cross through the membrane only one time. The process is the following one: - SRP (signal recognition particles) recognize the sequence and get in touch with the ribosome. - Transport the ribosome towards the membrane of the ERER. - Translation continues and when the protein is burning inside the lumen, the peptidase is gonna cut the signal sequence. - When the protein is finished the translocon closes again and we will have the protein. Also, the hydrophobic peptides will be transferred to the lipid bilayer. Alternating the hydrophobic sequences (start and stop) there is a possibility to generate multiple multipass of transmembrane protein. So the internal hydrophobic parts of the protein will constitute the transmembrane domains of the future protein. Hydrophobic amino acids tend to arrange alpha-helix 3D (most stable form of arrangement), which interact with the lipid bilayer (the amino acids are not repealed by the bilayer because they are hydrophobic, so they are able to integrate well in the lipid bilayer). *Non-well folded proteins can suppose a danger for the protein itself. If chaperons fail their function, then the protein won’t complete its function (not functional). (The function of each proteins relays on the 3D configuration of the protein) *These signal sequences get their name (start/stop) because the first sequence, which is the signal peptide, starts the translocation of the protein. But then, when there is another one, it will stop because the amino acid that is next to it will be sticking out of the membrane. 6.3. GLYCOSYLATION (No ha dicho todo) This process is specific for proteins taking the secretory synthesis pathway (RER-Golgi). It starts in the ER and it takes place simultaneously with the translocation. BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM During the translation of the protein some specific carbohydrates are added to it, because cytosolic proteins have no sugar residues attached. Those needed sugars are transferred from the main molecule used in this process: dolichol (phosphate). It is located in the ER membrane and has two functions: anchoring molecules and carrying molecules. As the proteins get synthesized, some specific amino acids of the proteins’ structure bind to the dolichol oligosaccharide, like this, the oligosaccharide is transferred from dolichol phosphate to asparagine amino acids in the new protein. This process is called N-glycosylation (carried out on the top in a nitrogen atom). Alway every oligosaccharides that take part contain the same elements (they are the same oligosaccharide): 14 sugar residues, 2 NAG (N acetylglucosamine), 9 mannose and 3 galactose. Apart from that, all the proteins that enter in the ER are glycosylated in the same way, initially. Particularly important in the golgi apparatus for tagging different proteins, due to the compounds taking different directions. 7. FOLDING AND QUALITY PROTEINS 7.1.CHAPERONS AND PROTEIN FOLDING Chaperons are proteins that help other proteins fold correctly, whether the type of the protein they all have to be fond correctly. Proteins that aren’t correctly folded are useless, and even dangerous for the cell (they become a problem for the cell). Due to that, and to help new proteins get their final 3D conformation, ER is very rich in chaperones. If chaperones fail, the new synthesized protein will likely be non-functional. One example of complex folding processes requiring the aid of chaperones is heteromeric protein assembly (antibodies), that need BIP chaperone. IgGs (a type of antibodies) contain two light and two heavy chains, so they are a protein containing four different protein chains. Here, BiP chaperones help them to stabilize, before they get bound to the rest. So this process takes place gradually. Another example is the case of the disulfide bond formation (covalent bond), in which the protein disulfide isomerase (PDI) has an important role: it participates in the creation of these specific unions by oxidizing amino acids with a sulfide group to create a covalent bond that brings polypeptide chains together. This is very typical in heteromeric proteins. *There are four main proteins that control the proper folding of proteins to manage to get that 3D structure, which are different types of chaperons: The following molecular chaperons are very abundant in the RER lumen. Calnexin and Calreticulin bind to glycosylated protein moieties, binding those oligosaccharides. BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM - BiP; It is hydrosoluble. It is a resident protein and it is found in the lumen in the ER. It stabilizes and checks the proper folding of the protein. - Calnexin; It is liposoluble because it is a transmembrane protein but it is facing the lumen so it can interact with the protein. It controls the proper folding of the protein by adding or removing sugars. - Calreticulin: It shares the function with the previous one but in this case it is hydrosoluble. PDI: It is hydrosoluble. It is found in the lumen of the ER and its main function is to create disulfide bonds. If a new proten is not well folded, it gets retained by chaperons inside the ER. 7.2 THE UNFOLDED PROTEIN RESPONSE (UPR) - ER STRESS: Folding and Quality control in the ER If chaperones manage to get proteins folded it happens what is called the unfolded protein response UPR. This is a situation of ER stress. Proteins need to be properly folded within the ER and if many unfolded proteins accumulate, that may compromise cell viability. There are some stress sensors (PERK, IRE1, ATF6) that are present in the ER membrane that can detect these situations. If you have a situation, for instance, when cells are subjected to high temperatures, many unfolded proteins are  obtained so there is a higher demand for chaperones. Chaperones instead of being free, they will be all occupied to fold the proteins. There are some transmembrane proteins (stress sensors) in the ER membrane which can be either bound to chaperones or unbound to them. When the chaperone demand is low, these proteins will be bound to chaperones in an inactive form. However, if there is a higher demand for chaperones, then those sensors will lose BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM the chaperon binding, and chaperones will bond to unfolded proteins. If there are a lot of unfolded proteins, the ER sensors don't have any chaperons bound to them and they activate the relay stress signals to the cell nucleus. This is what is called the UPR or the unfolded protein response. The important idea is that they all culminate in the activation of specific transcription factors that may affect gene expression. The gene expression response that is activated is usually to increase the chaperones synthesis as chaperones are very needed in the case of accumulation of unfolded proteins. Then, autophagy pathways will be activated as well. If this situation of ER stress persists, this will lead to the suicide of the cell, what we call as cellular apoptosis. This is usually created by the accumulation of unfolded proteins. 8. SMOOTH ENDOPLASMIC RETICULUM (SER) The SER is continuous with the RER and they both belong to the same organelle but the structure is completely different. In the SER there are not any attached ribosomes, so they cannot synthesize proteins. However, it is very important in lipid metabolism. Most cellular membrane lipids are synthesized here so they can then be transferred to other cellular compartments by means of vesicles or specific transporters. The SER also plays an important role in the clearance of toxic hydrophobic compounds (xenobiotics) by oxidating them. This is catalyzed by the p450 cytochrome system (redox enzyme system). They are very often hydrophobic so they need to be modified. Cells containing a very developed SER are usually involved in lipid metabolism and in the clearance of toxic substances in our organism. For example, liver hepatocytes (which are involved in the clearance of toxins or hydrophobic toxins from the blood, like in the drug clearance). Adrenal gland cells also have a very developed SER but in this case they are involved in steroid hormone synthesis. 8.1. CYTOCHROME P450 SYSTEM  Carries out the oxidation of the toxic compound Is a very powerful system to metabolize non-water soluble toxic organic compounds. Cytochrome p450 system retains a lot of mitochondrial reticulum transport chains. It is a set of redox enzymes present in the ER membrane with the function of oxidizing of organic components. There are electron donors (NADPH will be the primary donor) which transfer electrons to the chain of proteins to transport them from one another. This is used to metabolize organic compounds. These compounds are usually BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM hydrophobic toxins and in order to be eliminated/secreted they have to be oxidized. Those enzymes manage to combine the ‘R’ (this will refer to the toxin) to make it water soluble (more “manageable” for secretion/elimination). Similarly to mitochondria, this system consumes O2 that combines with the toxin so it gets oxidized and oxygen gets reduced to water. 8.2. MEMBRANE LIPID BIOSYNTHESIS IN THE SER The SER is also fundamental for lipid biosynthesis. This takes place in the cytosolic hemilayer of the SER membrane. New lipids will be added to the cytosolic hemilayer of the SER. In the image we can see the process of phospholipid synthesis (the most abundant lipid in our membranes). This is formed by the esterification of fatty acids with glycerol thanks to acid transferases which are present in the SER membrane. These acid transferases generate phosphatidic acid at the beginning, then the phosphate group gets removed and the diacylglycerol, which is the product of the removal, can then be combined with nonpolar groups in different types of phospholipids. In the case of the choline combination, you get phosphatidylcholine. 9. INTEREXCHANGE OF LIPIDS BETWEEN THE ER AND OTHER ORGANELLES  We know that phospholipids can flip to the opposite hemilayer and then they can diffuse laterally; they can travel. Phospholipids in the ER get transported to the GA by means of vesicles (this is the typical way of transport). The addition of new phospholipids takes place only in the cytosolic hemilayer of the SER membrane, but some of them will then move to the luminar part hemilayer, to equilibrate both sides. Those phospholipids will flip (flip-flop movement) inside. Once synthesized they can transfer to other organelles by means of vesicles or transporters. Sphingolipids, however, are a bit different because the basic block of sphingolipids is ceramide and it doesn’t travel in vesicles. This is transferred through specific translocators which bring these ceramides directly from the SER membrane to the golgi apparatus. They get sphingolipids BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM such as sphingomyelin and once they have mature sphingomyelin, this goes together with phospholipids in secretion vesicles to reach the plasma membrane. ER may transfer lipids directly to other membrane organelles like mitochondria and peroxisomes, even the plasma membrane. They are lipids of the nuclear envelope, which can diffuse laterally. Since the nuclear envelope is continuous with the ER, the destination is reached. As you can see, there are many ways to transfer lipids from the ser to different membrane compartments: direct, through vesicles, specific translocators… 10. OTHER ER FUNCTIONS: STORAGE OF Ca2+ The endoplasmic reticulum can also store Ca2+ from the cytosol and release it in response to specific signals. It is able to transport it against gradients (active transport, calcium pump). When looking at the calcium concentration in the intracellular and extracellular medium, we can see that the extracellular medium contains high concentration of calcium. The cytosol, in contrast, contains a very low one. But the lumen of ER is very rich in calcium as well, and this is so because in the membrane of the ER we have some transmembrane proteins which hydrolyze ATP to keep calcium released from the ER. This regulates a lot of physiological processes, for example, muscle contraction. Many signaling pathways culminate in the release of calcium from the ER. This is the case of skeleton muscles. Most of the calcium that overwhelms the cytosol upon muscle fiber contraction comes from the ER, and not from  the intracellular space. This is just one example of the many different processes that can be regulated by calcium released from this organelle. KNOWLEDGE TEST: 1. Why is the ER divided into smooth and rough ER (SER, RER)? Because the rough one has ribosomes attached to it and the smooth has not. 2. Which are the main functions of each two parts? The RER is involved in protein metabolism: protein synthesis (beginning of secretory pathway), protein sorting, protein glycosilation and protein folding while SER is involved in lipid metabolism: membrane lipid synthesis, interexchange of lipids between organelles, cholesterol and steroid hormone synthesis and detoxification of organic compounds. 3. Proteins synthesized in the ER have a keysignature/specific characteristic that is not found in proteins synthesized in the cytosol. Which is it? Glycosylations, which involve adding sugar molecules. This process distinguishes them from cytosolic proteins, which do not undergo glycosylation. BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM 4. What is it needed for a protein to be translocated to the ER? A protein needs a signal peptide, which is a sequence of 8-10 hydrophobic amino acids near the start, to be moved to the ER. This signal starts the process and is recognized and attached by the Signal Recognition Particle (SRP). SRP is located in the citosol, in the very beggining of the protein. 5. The lumen of the RER is very rich in molecular chaperones. What is the function of those proteins? Can you name some examples of them? Chaperons are proteins that assist in the correct folding of other proteins so they get their final 3D configuration. For example, BIP proteins. 6. What is the ER stress respone? Is the reaction that the ER transmit to the nucleus if there are too many misfolded proteins accumulated in the ER. This response can lead to block the expression of more proteins or even be the cause of the suicide of the cell. 7. How does the SER participate in the clearence of toxic compounds? By the cytochrome p450 system, who metabolizes non-water soluble toxic organic compounds by oxidating them so they become non polar. 8. The SER can transfer membrane lipids to other eukaryotic cell compartments. Can you briefly describe the different mechanism to do that? They can travel to the membrane through vesicles and by specific trannsolcator (those mainly when the molecules have to be transported to the GA) 9. How does the ER sequester Ca+2 to attain a higher concentration of this ion inside the ER lumen? ER collects Ca2+ from the cytosol, increasing its levels inside. It can release this Ca2+ when signaled to do so. This is due to the presence of pumps that pump calcium against the gradient concentration. Ca2+ in the cytosol is important for many cell functions, including muscle contraction. 10. What is the main function of signal sequences and SPR in rotein synthesis on the ER? Facilitate ribosome binding to the ER membrane 11. Membrane-bound proteins are integrated into the ER membrane because they posses: Transmembrane hydrophobic domains. 12. The Unfolded Protein Response (UPR) aims to restore ER homeostasis by: Enhancing protein synthesis BIOCELL BOCK 4: ENDOMEMBRANE SYSTEM 13. In addition to lipid synthesis the smooth endoplasmic erreticullum is also involved in the metabolism of which type of molecule? Xenobiotics (foreign organic compounds)