Golgi Apparatus (Corrected) PDF

1st Medicine Cellular Biology Group 7 TOPIC 8: THE GOLGI APPARATUS (BLOCK 4: THE ENDOMEMBRANE SYSTEM) 1. The apparatus 1.1.Discovery 1.2.Structure 1.3. Functions 1.4.Movement of con...

1st Medicine Cellular Biology Group 7 TOPIC 8: THE GOLGI APPARATUS (BLOCK 4: THE ENDOMEMBRANE SYSTEM) 1. The apparatus 1.1.Discovery 1.2.Structure 1.3. Functions 1.4.Movement of contents across the GA 2. Protein glycosylation in the GA. 2.1. N- and O- glycosilations 2.2.GAG and PG synthesis in the GA 3. Controlling the final destination of proteins 4. Bidirectional communication between ER and Golgi 5. Lipid biosynthesis in the Golgi complex 1. The apparatus 1.1.Discovery The Golgi apparatus is a very important part of the endomembrane systems. Its name comes from Camillo Golgi, which discovered it in 1898. He developed a particular technique (the silver impregnation technique), which consists in the precipitation of specific metals (silver metals) in the areas which are rich in the golgi apparatus to stain them. In some Spinal ganglion neuro dark spots show silver precipitates (the Golgi Apparatus). 1.2.Structure The Golgi apparatus is a stack of quite flat systems which are close to each other but not connected. The GA is the director of vesicle trafficking. The GA receives contents from the 0  1st Medicine Cellular Biology Group 7 ER in the form of vesicles and those vesicles fuse with the CIS side of the GA. Then they are delivered into more specific vesicles of the TRANS side. By the time the products get out of the cell they are classified according to their final destination. It is a polarized organelle, which means that it has 2 sides: the CIS side and the TRANS side. Watching the GA by TEM, since they are a little bit curvated, we can difference them because the CIS side is convex, while the TRANS side is concave. By the time vesicles and products go out of the TRANS side, they are sorted out in different vesicles due to their sugar pattern. This sugar pattern is distinguishable and identified by different cells. By TEM we can see how the CIS side is convex and the TRANS concave. 1  1st Medicine Cellular Biology Group 7 The golgi apparatus can be also called “Golgi complex”. As mentioned before, it is created by various compartments or cisternae, which are independent from each other, but they are piled very tightly together. The inside of these cisterns is called lumen. Besides, the golgi apparatus will receive vesicles which come from the ER (endoplasmic reticulum). In the inside of the apparatus, those vesicles will have to cross those cisternae while some reactions take place, helping the sorting and classification of the products into different vesicles according to their final destination. Each cistern has a different glycosyl transfer reaction. Basically the GA is a giant sugar transferring machine, because it adds sugar residues to different proteins.We can see which enzymes are in each of the compartments. 1.3. Functions The GA participates in: - Lipid and glycolipid biosynthesis (particularly in sphingolipid synthesis) - Protein glycosylation and modification - Lysosomal biogenesis - Sorting of cargo proteins into different vesicles The GA has 2 sides (CIS and TRANS) and has different functions. The CIS side is the receptor of the products that come to the GA and the TRANS side is the “expulsor”, which means that products leave the GA from that side. Products will have to go through the different cisternae of the GA. The products which enter the apparatus from the CIS side are not classified (the side where products are incorporated is called Cis-Golgi Network or CGN). However, vesicles which come out of the TRANS area (the part from which vesicles leave the GA is the Trans-Golgi Network or TGN) are classified into different vesicles thanks to the reactions which take place in the different compartments of the apparatus and of the vesicles that get out contain a glycosin transport. This is why GA can also be called director of intracellular vesicle trafficking, because thanks to the reactions which take place there, vesicles will be classified depending on their final destination. 2  1st Medicine Cellular Biology Group 7 The most typical changes that take place in the GA to achieve the classification are glycosylations. So we can say that proteins are heavily glycosylated here. Products which contain similar glycosylation patterns, will be stored in the same TGN vesicles, and in consequence, will have the same destination. There are 3 main destinations (TGN released vesicles): Secretory vesicles: ○ Constitutive secretion: constantly exocyted without any control. ○ Regulated secretion: vesicles are stored in the cytoplasm and will wait to be released. This release will only occur when the cell receives the signal to do so, such as neurons. Lysosomes: membrane compartment vesicles that contain high levels of hydrolytic enzymes which contain the mannose-6-phosphate, so that they can carry out digestion in lysosomes. Retrograde transport vesicles: they go back to the CIS golgi network and to the ER to maintain the proteins which fulfill their function in the ER. Golgi apparatus is compartmentalized. This means that in each cisternae some sequential enzymatic reactions will take place. So there are different enzymes located in different cisternae. So we must bear in mind that products leaving the TGN are already sorted (or classified) according to their final destination. 3  1st Medicine Cellular Biology Group 7 Once translation occurs in the ER, the proteins don't stay there, they must undergo the last changes in the golgi apparatus. So once the protein has been properly modified, it will go back to the ER. 1.4. Movement of contents across the GA There are 2 mechanisms which coexist: 1. CISTERNAL MATURATION MODEL: the GA is constantly receiving new membranes from the ER. Like this, intermembrane compartments between the ER and the GA are created. These new membranes will fuse to the CIS part of the GA. The old membranes will be pushed out. Cisterns will mature as they are “pushed” from the CIS to the TRANS side. New membranes are coming and leaving the GA all the time. This has implications. As each cistern contains a specific set of proteins(enzymes). As new membranes enter the GA in order to keep their position, those proteins(enzymes) need to be retrotransported from one compartment to another. The enzymes will have to undergo retrograde transport, as they are also being pushed forward by specific vesicles to be transported back to stay in place. 2. VESICLE TRANSPORT MODEL: Vesicles are created and bound at the edges of the GA. They move contents back (anterograde) and forth (retrograde) by those lateral vesicles. They can move from the TRANS side to the CIS side, or vice versa. If there was no lateral transportation, contents would eventually get out as new membranes push. ➔ BOTH MODELS COEXIST 4  1st Medicine Cellular Biology Group 7 2. Protein glycosylation in the GA Glycosylations that occur in the GA have already started in the ER, but the GA largely modifies the glycosylation of proteins. The proteins which enter the CGN will all have the same oligosaccharide (14 residues) attached to Asn aminoacids. But only the central part will remain (5 residues: 2 N-acetyl glucosamine and 3 manoses). Once in the GA, this central oligosaccharide will be lastly modified. The Asn residues can be glycosylated following 2 different patterns: - Complex glycosylation: it consists of the addition of varied sugars to the central part (for example, galactose, N-acetyl glucosamine…). Depending on the oligosaccharide which is added, the protein will have one destination or another different one. But most of the proteins that are modified in this way will take the secretion pathway, so they will be membrane and secretion proteins. Highly variable and specific to the protein. - Mannose glycosylation: only mannose monomers are added to the central part. When these mannose residues are phosphorylated, this will constitute the tag or labeling to bring them to lysosomes, so they will be future lysosomal proteins (characteristic of lysosomal proteins and then this mannose will be phosphorylated). 2.1. N- and O- glycosylations Glycosylations can happen in two ways, and both can happen in the Golgi Apparatus: - N glycosylations: sugars residues are added to the nitrogen atom on asparagine amino acids. This glycosylation pattern is shared with the ER. In fact, it is started in the ER but then, these oligosaccharides are lastly modified in the GA. - O glycosylations: (highly specific of the GA) sugar residues are added to the O atom of serine and threonine amino acids. This process is exclusive of the GA, so it begins and ends there. 5  1st Medicine Cellular Biology Group 7 2.2. GAG and PG synthesis in the GA By adding sugars, the GA synthesizes essential components of the Extracellular Matrix. These are secreted by constitutional secretion. Between these components we have Glycosaminoglycans (GAG), which are long chains of disaccharides repeated hundreds or thousands of times. These molecules are polar, so they are hydrophilic and can be charged. This is a representation of a GAG molecule GAG can also be bound to proteins by O glycosylation, and in this way, they create proteoglycans. Finally, proteoglycans (PG) are very big molecules. They consist of a protein core with many GAGs bound to it. This is a drawing of PG. They are all heavily glycosylated. 6  1st Medicine Cellular Biology Group 7 3. Controlling the final destination of proteins Proteins which exit the TGN (Trans Golgi Network) as vesicles and have 3 different main destinations: LYSOSOMES: hydrolytic enzymes which are rich in phosphorylated mannose will go there. SECRETION VESICLES: ○ CONSTITUTIVE SECRETION VESICLES: The constitutive secretory pathway is the default pathway. They are targeted directly to the plasma membrane and there is no control of it, so they need no signal. They are constantly produced and get out of the cell. There are parts of transmembrane proteins that are facing the membrane of the vesicle that will end up facing the outside. When this vesicle fuses with plasma membrane the transmembrane proteins that were facing will turn and face out. ○ REGULATED SECRETION VESICLES: they also contain specifically sorted products but they will be stored in the cytoplasm waiting for the release-signal. When this appears, this will trigger the fusion of these vesicles with the plasma membrane to release these contents all at once. Some of them are mannose-6-phosphate receptors. RETROGRADE TRANSPORT VESICLES: vesicles go back to the ER. TGN will classify each protein according to their final destination thanks to specific tags that have been placed in the proteins in the different systems of the GA. In the case of lysosomal enzymes, these signals or tags are Mannose-6P. M6P receptors are facing the luminal part of the TGN and will bind only to proteins containing mannose 6P. Then, thanks to some specific coating proteins (clathrins) lysosomal vesicles will be created. *mannose must be phosphorylated (creating Mannose-6P) 7  1st Medicine Cellular Biology Group 7 In this picture we can see M6P receptors which are facing the lumen of the TGN are only binding to proteins which contain Mannose 6P (YELLOW). We can also see proteins leaving the TGN which will follow the regulated secretion and will be stored in the cytoplasm until the release signal arrives (PINK) and proteins which will follow the constitutive secretory pathway and are directly leaving the cell (GREEN). In the case of I cell disease, the mannose residue doesn't get properly phosphorylated. So then, hydrolytic enzymes do not get correctly sorted out and they get released out of the cell. This leads to the generation of some severe syndromes, basically because hydrolytic enzymes are not properly driven to their destination (lysosomes). LYSOSOMAL BIOGENESIS IN THE TGN The signal for lysosomal sorting is added in the CGN, there happens the addition and phosphorylation of mannose, then the recognition of this signal is performed in the TGN, where proteins which are rich in mannose-6-phosphate bind to their specific receptors which are concentrated on specific sites of the TGN. It is important to know that those receptors will be segregated to specific parts of the TGN. Clathrin also assists the process of creating vesicles. Many hydrolytic enzymes (vesicles) will fuse with each other to create primary lysosomes, so the lumen of lysosomes will be acidic. Due to the acidification of the lysosomal lumen (because of the proton pumps), the release of the ligand and the receptors happens. So ligands, which are hydrolytic enzymes, will be concentrated in the lysosomal lumen, whereas receptors will be recycled back to the TGN to participate in the recruitment of more hydrolytic enzymes. So in conclusion, we can say that the creation of lysosomes all depends on the concentration of lysosomal enzymes, as well as mannose-6-phosphate tags and MP6 receptors. In this picture the process of the creation of a primary lysosome is graphically explained. 8  1st Medicine Cellular Biology Group 7 4. Bidirectional communication between ER and Golgi As mentioned before, retrograde transport of products from GA to ER is possible. There will be some vesicles that will go through the whole GA back to the ER (retrograde transport vesicles), and those vesicles will be transporting ER resident proteins. So, proteins that get to the ER as their final destination, are not synthesized and stay there for the whole time, they have to go to the GA, and then, they will have to be retrieved back to the ER. We have 2 pathways: - Forward pathway or anterograde transport: consists of transport from the ER to the Golgi. Vesicles which take part in this transport are called COPII vesicles. They contain a specific coat (COPII) and go from the ER to the GA. - Retrieval pathway or retrograde transport: consists of transport from the GA back to the ER. The vesicles which take part are COPI vesicles. This is the case of ER resident proteins. The retrieval of proteins is mediated by KDEL receptors in the TGN. Proteins that need to be retrieved to the ER have a specific signal which consists of a short strand of 4 amino acids (Lys-Asp-Glu-Leu). This way COPI vesicles will identify the signal and take the protein back to the ER. The role of COPI and COPII is to facilitate vesicle formation. Yellow ones are secretory proteins and pink ones are ER resident proteins. Both proteins get together in the COPII vesicles to the CIS Golgi part, but then in the GA, secretory vesicle will be recognised and sent out by constitutive or regulated secretion. And ER proteins will be recognised by specific receptors that are facing the lumen of the TGN. Those receptors are called KDEL receptors. ER resident proteins will have a tag (KDEL) which consists of some short strands of 4 Aa which are Lys-Asp-Glu-Leu (KDEL). This KDEL signal will be exclusively in proteins which make part of the ER. Then, COPI vesicles containing KDEL receptors will bring these vesicles back to the ER from the TGN 9  1st Medicine Cellular Biology Group 7 5. Lipid biosynthesis in the Golgi complex The last steps of the biosynthesis of Sphingolipids takes place in the GA. So we must remember that sphingolipids are created by a ceramide and different polar groups. The ceramide is synthesized in the SER and gets translocated to the GA thanks to specific transporters. This ceramide does not travel in vesicles, it gets translocated specifically. Once in the GA, this ceramide may undergo 2 types of modifications. The first modification would be to add sugars to it and generate glycolipids; and the second one is to add phosphocholine creating sphingomyelin, which together with the phosphatidylcholine, are the most abundant lipids in our membranes. KNOWLEDGE TEST 1. Why do we say that the GA is a polarized organelle? We say that the GA is a polarized organelle because morphologically it is divided in two different sides: CIS and TRANS sides. The CIS side receives vesicles from the ER, while the TRANS side releases vesicles from the GA. The flow of materials from the cis to trans side, and the differential composition of enzymes in each part of the GA, contribute to its polarized nature. 2. How do contents cross through the different cisternae of the GA from the CIS to TRANS side? 10  1st Medicine Cellular Biology Group 7 Movement of contents across the golgi apparatus: 1) Cisternal maturation model. The Golgi apparatus constantly receives new membranes from the endoplasmic reticulum. These fuse with each other and form the cis side of the GA. The entering of new vesicles or membranes in the GA will make the others mature and be pushed away. Cisterns mature as they are “pushed” from CIS to TRANS side. During this maturation process, the cisterna undergoes enzymatic changes, acquiring and losing enzymes needed for specific modifications at each stage. 2) Vesicle transport model. Proteins are packaged into small vesicles that bud off from one cisterna and fuse with the next cisterna in the stack. This allows the cargo to move from the cis-Golgi to the trans-Golgi. 3. What are the possible destinations of vesicles exiting the GA? The destinations of vesicles exiting the GA could be secretory vesicles (constitutive secretion or regulated secretion), lysosomes or retrograde transport vesicles (from Golgi back to ER). The release mechanism is different according to the destination. 4. How does the GA recognize and sort products according to their destination? Sugars are going to be added to the proteins by glycosylation patterns. The different glycosylation patterns (sugars added) will be recognized. Sugars will be recognized by specific receptors in the lumen of GA. Proteins having specific glycosylation patterns will make the proteins be transported to specific parts of the transport network because of mannose binding receptors, specifically mannose 6-P. So, everything relies on glycosylation patterns and the specific binding of those sugars to the receptors that we find in the lumen of the GA. 5. The GA is essential for the synthesis of several components of the extracellular matrix (ECM). Can you give some examples? Proteoglycans (glycosaminoglycans bound to proteins) and glycosaminoglycans (long chains of disaccharides). Both are important components of the extracellular matrix which are synthetized in the GA. 6. Some types of important membrane lipids end their synthesis in the GA. Which ones? Sphingolipids (ceramides come from the ER and in the GA are formed the sphingolipids) and glycolipids where sugar residues are added. 7. What is the signal to sort protein contents from the GA to lysosomes? The key signal for targeting proteins to lysosomes is the mannose-6-phosphate (M6P) tag. This tag is added to proteins in the Golgi, and the mannose-6-phosphate receptors in the trans-Golgi recognize and sort these tagged proteins into vesicles destined for the lysosomes. 8. And to bring proteins from the GA back to the ER? KDEL receptors (small peptide, 4x aa = Lys-Asp-Glu-Leu). The GA will pack them into vesicles for retrograde transport to the ER. Basically, it is a peptide formed by 4 amino acids that are present in 11  1st Medicine Cellular Biology Group 7 the proteins that need to go back from the GA to the ER. Those specific proteins are recognized by receptors in the lumen. 9. What is the fate of products that show no specific sorting signal in the TGN? Products that lack a specific sorting signal in the trans-Golgi network (TGN) are typically sent to the default pathway, which generally leads to constitutive secretion (out of the cell). ➔ Constitutive Secretion (default pathway): Continuous and unregulated, delivering products to the plasma membrane or extracellular space. What is the primary function of the GA? a) Protein synthesis b) Lipid storage c) Protein degradation d) Protein modification and packaging Which of the following describes the structure of the Golgi apparatus? a) Stacks of flattened membrane sacs b) Long, thread-like extensions c) Smooth, tubular structures d) Spherical organelles What is the main role of the GA in the production of carbohydrates? a) Synthesizing glucose for energy production b) Modifying and adding sugar residues to glycoproteins and glycolipids. c) Breaking down complex carbohydrates into simple sugars. d) Storing glycogen for later use. What is the primary function of receptors located in the trans -golgi network? a) Exporting proteins from the GA b) Sorting and targeting proteins to their final destinations c) Synthesizing lipids for cellular membranes d) Breaking down cellular waste products. Which of the following correctly describes constitutive secretion? a) It is triggered by specific signals, such as hormone binding b) It occurs continuously without the need for external signals c) It involves the recycling of vesicles back to the GA. d) It delivers enzymes directly to the lysosome In regulated secretion, vesicles remain in the cytoplasm until which event occurs? a) The vesicles receive a signal, such as an increase in intracellular calcium levels 12  1st Medicine Cellular Biology Group 7 b) They are degraded by lysosomes c) The Golgi apparatus releases them automatically d) They fuse with lysosomes for maturation 13