Chapter 8: An Overview of the Endomembrane System PDF

CHAPTER 8 An Overview of the Endomembrane System  Membranes divide the cytoplasm of eukaryotic cells into distinct compartments.  The endomembrane system consists of the nuclear envelope and organelles such as the endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and vacuoles functionin...

CHAPTER 8 An Overview of the Endomembrane System  Membranes divide the cytoplasm of eukaryotic cells into distinct compartments.  The endomembrane system consists of the nuclear envelope and organelles such as the endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and vacuoles functioning as part of a coordinated unit. What about mitochondria and chloroplast and peroxisomes? Why? Fig 8.1: Membrane-bound compartments of the cytoplasm An Overview of the Endomembrane System • Organelles of the endomembrane system are part of an integrated network in which materials are shuttled back and forth. • Materials are shuttled between organelles in membrane-bound transport vesicles. • Upon reaching their destination, the vesicles fuse with the membrane of the acceptor compartment. Fig 8.2A: Inside vesicle: orientation remains the same © 2013 John Wiley & Sons, Inc. All rights reserved. Question Which ER is involved in protein production? Why? Rough ER Studded with ribosomes Involved in protein production Smooth ER Devoid ribosomes Associated with lipid manufacture and metabolism and steroid production © 2013 John Wiley & Sons, Inc. All rights reserved. Overview of the Endomembrane System Biosynthetic and secretory pathways  Several distinct pathways through the cytoplasm have been identified. • Biosynthetic pathway – synthesis, modification and transport of proteins. • Secretory pathway – when proteins are discharged (secreted) from the cell.  Constitutive secretion – in a continuous fashion.  Regulated secretion – in response to a stimulus. Endocytic pathways that unite endomembranes into a dynamic, interconnected network. Fig 8.2B © 2013 John Wiley & Sons, Inc. All rights reserved. Overview of the Endomembrane System Biosynthetic and secretory pathways Natalia HR et al., Journal of Cell Science 2020 A stimulus upregulates transcription/translation of a factor that is trafficking to the plasma membrane resulting in the continuous secretion. Replenish the material at the plasma membrane and certain membrane bound organelles Example: Fibroblasts constitutively secrete proteins like Collagen and Proteoglycans into the extracellular matrix and play an important role in maintaining the structural integrity of connective tissues. Protein accumulate in storage granules and upon arrival of stimulus storage granules fuse with the plasma membrane resulting in exocytosis. Release the material when stimulus is there. Example: secretion of digestive enzymes by pancreatic cells Overview of the Endomembrane System How can we study endomembrane system? Electron microscopy has provided a detailed portrait of the structure of the cells. Some approaches/methods used to study are 1. Autoradiography 2. GFP- based protein tracking 3. Biochemical analysis of Subcellular Fractions 4. Use of Cell-Free Systems 5. Utility of genetic mutants © 2013 John Wiley & Sons, Inc. All rights reserved. An Overview of the Endomembrane System Pathways secretory Biosynthetic constitutive regulated Ways to study endomembrane system- 5 ways 1. Autoradiography 2. GFP- based protein tracking 3. Biochemical analysis of Subcellular Fractions 4. Use of Cell-Free Systems 5. Utility of genetic mutants Study of Endomembranes 1. Autoradiography Insights Gained from Autoradiography  Autoradiography – a method to visualize biochemical processes using radiolabeled materials exposed to a photographic film.  Can be used to determine where secretory proteins are synthesized by using labeled amino acids.  Techniques utilizing autoradiography have been largely supplanted by fluorescent-based approaches. Fig 8.3a: Pancreatic acinar cell incubated 3 min with ‘hot’ amino acids. Silver grains localized over the ER © 2013 John Wiley & Sons, Inc. All rights reserved. Study of Endomembranes 1. Autoradiography: Synthesis and transport of secretory proteins Fig 8.3b-d 3 minute pulse: radioactively labeled amino acid (red) • • • • • • 3 minute pulse: 17 minute chase 3 minute pulse: 117 minute chase Radioactive labelled amino acids were introduced into the cell Cell will stat making proteins by using the radioactive labelled amino acids (3 minutes pulse) A non radioactive labelled amino acid was introduced in the cell (for 117 minutes as a chase) Fig B shows that radioactivity is localized in the ER Fig C shows that radioactivity is in the Golgi complex and the secretory granules Fig D shows that radioactivity is in the secretory granules and is begin to released into the pancreatic ducts Why different time points were tested? To see the newly synthesized molecule in the cytoplasm (more time more travelling of the newly synthesized molecule) https://www.youtube.com/watch?v=i_symXB5KUw Study of Endomembranes 2. GFP-based protein tracking Use of Green Fluorescent Protein  Green fluorescent protein (GFP) – a small protein isolated from jellyfish which emits green fluorescent light.  A GFP-DNA chimera allows to observe the protein synthesis in the cell.  Fusing viral genes to GFP allows the study of protein traffic due to large production of proteins. https://blog.microbiologics.com/w p-content/uploads/2016/08/1010-768x1152.jpg protein stuck in ER  The use of green fluorescent protein (GFP) reveals the movement of proteins within a living cell. Fun Fact Immortalized jelly fish (Turritopsis dohrnii) proteins can move from ER to Golgi Study of Endomembranes 3. Biochemical Analysis of Subcellular Fractions Sub-cellular Fractions  Techniques to homogenize cells and isolate some organelles, which can then be separated from one another through subcellular fractionation.  Membrane vesicles derived from the endomembrane system form a collection of vesicles called microsomes, which can be characterized further through other techniques e.g. proteomic technology by mass spectrophotometry. Smooth ER-derived Rough ER-derived Fig 8.5a Isolation of a microsomal fraction © 2013 John Wiley & Sons, Inc. All rights reserved. Study of Endomembranes 3. Biochemical Analysis of Subcellular Fractions Smooth ER-derived Microsomes: (Natural or artificial)?- Why we need it? Artifical and commonly used to investigate the structural and functional aspects of the ER  Microsomes are invaluable tools to study the enzyme inhibition, rates and efficiency of clearance, metabolite identification or drug-drug interaction (invitro study) Rough ER-derived Fig 8.5a https://www.beckman.com/resources/sample-type/extracellular-vesicles/microsomes Isolation of a microsomal fraction Study of Endomembranes 4. Use of Cell–Free Systems Use of Cell-Free Systems  Cell-free systems do not contain whole cells and have provided information about the roles of the proteins involved in membrane trafficking.  Other type of information identified includes: proteins that bind to the membrane to initiate vesicle formation, and proteins responsible for cargo selection.  James Rothman and Randy Schekman got Nobel prize for their use of cell-free system in their research on vesicle trafficking. Fig 8.6: Formation of coated vesicles in a cell-free system  Figure shows a liposome with vesicles budding from its surface. Buds and vesicles were produced when prepared liposome was incubated with purified proteins.  This shows that cellular processes are reconstituted in-vitro from the purified components. © 2013 John Wiley & Sons, Inc. All rights reserved. Study of Endomembranes 5. Utility of genetic mutants Study of Mutants  Mutants provide insights about the function of normal gene products.  Isolation of proteins from yeast has led to the identification of homologous proteins in mammals, pointing to the conserved nature of endomembrane systems. Fig 8.7: Use of genetic mutants in the study of secretion Fig 8.7 Cross section of a wild-type yeast cell Mutation in a gene that encodes involved in the formation of vesicles Mutation in a gene that encodes involved in vesicle fusion The Endoplasmic Reticulum  The endoplasmic reticulum (ER) comprises a network of membranes that penetrates much of the cytoplasm.  Like other organelles, the ER is highly dynamic undergoing continual turnover and reorganization.  Divided into two sub-compartments: – Rough endoplasmic reticulum (RER) & Smooth endoplasmic reticulum (SER) – In addition, the composition of the luminal or cisternal space inside ER membranes is different from the surrounding cytosolic space. Electron micrograph: rough ER Rough ER in a pancreatic acinar cell Electron micrograph: SER Fig 8.8a,b smooth ER Leydig cells from the testis showing extensive smooth ER (steroid hormones synthesis) The Endoplasmic Reticulum Smooth Endoplasmic Reticulum (SER)  Extensively developed in a number of cell types such as skeletal muscles, kidney tubules and steroid-producing endocrine glands. SER functions include:  Synthesis of steroid hormones in endocrine cells: endocrine cells of the gonad and adrenal cortex.  Detoxification in the liver of various organic compounds (barbiturates and ethanol)  Detoxification is carried out by a collection of oxygen transferring enzymes (oxygenases) including the cytochrome P450 family (home of the P450 enzymes). These enzymes have lack of substrate specificity and able to oxidize thousands of different hydrophobic compounds and convert them into more hydrophilic excreted derivatives. Problem A harmless compound benzo[a] pyrene produced when meat is charred on grill and liver converts into a potent carcinogen by the detoxifying enzymes. Also metabolize many prescribed medicines Fig 8.10:Leydig cell: extensive SER where steroid hormones are synthesized Sequestration of calcium ion from cytoplasm of muscle cells: contains a high concentration of calcium-binding proteins. The Endoplasmic Reticulum-RER Rough Endoplasmic Reticulum (RER):  Composed of a network of flattened sacs (cisternae).  Continuous with the outer membrane of the nuclear envelope and also has ribosomes on its cytosolic surface.  Different types of cells have different ratios of the two types of ER, depending on activities of the cell. Fig 8.9 a: Schematic diagram showing stacks of flattened cisternae that make up the rough ER Fig 8.9 b: Transmission electron micrograph of Fig 8.9 c Immunofluorescence for the the rough ER in pancreatic acinar cells ER enzyme protein disulfide isomerase © 2013 John Wiley & Sons, Inc. All rights reserved. The Endoplasmic Reticulum-RER Functions of the RER a)Synthesis of Proteins on Membrane-Bound versus Free Ribosomes  Approximately one-third polypeptides encoded by the human genome are synthesized on ribosomes of RER and released into the ER lumen in a process called co-translational translocation.  It include secreted proteins, integral membrane proteins, and soluble proteins of organelles that resides in the endomembrane system (ER, Golgi complex, lysosomes, vesicles, plant vacuoles)  Polypeptides synthesized on “free” ribosomes and released into the cytosol.  It include: cytosolic proteins (glycolysis enzymes, proteins of the cytoskeleton), peripheral membrane proteins, nuclear proteins, and proteins incorporated into chloroplasts, mitochondria, and peroxisomes. © 2013 John Wiley & Sons, Inc. All rights reserved. The Endoplasmic Reticulum-RER What determines the site of synthesis of protein?  Site of synthesis of a protein determined by sequence of amino acids in N-terminus. a) A signal sequence at N-terminus attaches to secretory proteins that directs the proteins and ribosomes to the ER membrane. a) Polypeptide moves into ER cisternal space through a protein-lined pore, an aqueous channel in the ER membrane. The movement of polypeptides through the membrane as it is being synthesized is called (cotranslationally) or post-translational. © 2013 John Wiley & Sons, Inc. All rights reserved. The Endoplasmic Reticulum-RER a)Synthesis of a protein on membrane-bound ribosome Fig 8.12: A schematic model of the synthesis of a secretory protein (or a lysosomal enzyme) on a membrane-bound ribosome of the RER © 2013 John Wiley & Sons, Inc. All rights reserved. The Endoplasmic Reticulum-RER a)Synthesis of protein on membrane-bound ribosome Steps involved in the synthesis of Lysosomal protein on Membrane-Bound Ribosomes  Messenger RNA binds to free ribosomes on cytosol.  Secretory proteins synthesized on membrane-bound ribosomes have their signal sequence recognized by a signal recognition particle (SRP)  SRP-ribosome complex binds to an SRP receptor situated within the ER.  Attachment to the SRP receptor releases SRP  Newly synthesized protein moves to the translocon (a protein channel embedded in the ER membrane through which it moves to the ER lumen)  Upon entering the RER lumen, the N-terminal portion containing the signal sequence is cleaved by a proteolytic enzyme, signal peptidase.  Then, carbohydrates are added by the enzyme oligosaccharyltransferase.  The RER lumen is packed with chaperones to assist in folding, and also contains protein disulfide isomerase to add disulfide bonds to cysteines.  BiP (Binding immunoglobulin protein) is a molecular chaperone located in the lumen of the ER and binds to the newly synthesized proteins that are translocated into the ER (help in folding and oligomerization) © 2013 John Wiley & Sons, Inc. All rights reserved. The Endoplasmic Reticulum b)membrane biosynthesis in the ER Membrane Biosynthesis in the ER  Membranes arise from preexisting membranes.  Lipids are inserted into existing membranes.  As the membrane moves one compartment to the next, its proteins and lipids are modified.  Membrane asymmetry is established initially and maintained during trafficking. Fig 8.14: Maintenance of membrane asymmetry © 2013 John Wiley & Sons, Inc. All rights reserved. The Endoplasmic Reticulum c) Synthesis of Membrane Lipids  Most membrane lipids synthesized within the ER except sphingomyelin and glycolipids, and some unique lipids of mitochondria and chloroplasts.  Newly synthesized phospholipids are inserted into half of bilayer facing the cytosol, and then flipped into opposite leaflet by flippases.  There are enzymes that modify lipids already present within a membrane. Fig 8.15: Histogram indicating percentage of each of three phospholipids in three different cellular membranes- shows that the %age of each lipid changes gradually as membrane flows from the ER to the Golgi complex © 2013 John Wiley & Sons, Inc. All rights reserved. The Endoplasmic Reticulum Quality control: ensuring that misfolded proteins do not proceed forward. What happens to the misfolded protein? Mechanisms that ensure the destruction of misfolded proteins  Misfolded proteins are not destroyed in the ER; instead they are transported into the cytosol where they are destroyed in proteasomes.  This process is called ER-associated degradation (ERAD), and ensures the misfolded proteins do not reach the cell surface.  Proteasomes are protein complexes which degrade unneeded or damaged protein by proteolysis.  Lysosome contains digestive enzymes and break down the wornout cell parts. Pisoni et al., 2016. Traffic Interchange The Endoplasmic Reticulum Quality control: Proteasome Pathway  Ubiquitin (Ub) is a small regulatory protein that direct the movement of protein in the cell.  Ubiquitin molecules attached to the protein substrate  Recognized by 26S proteasome  Ub is removed and protein is linearized and injected into the central core of the proteasome and digested to peptides https://jasn.asnjournals.org/content/17/7/1807 Question: What is NOT true for SER? a) Synthesis of proteins b) Synthesis of lipids of a cell’s membrane c) Synthesize phospholipids and cholesterol d) Site of steroid hormone synthesis © 2013 John Wiley & Sons, Inc. All rights reseved. The Golgi Complex Fig 8.20: Schematic model of a portion of a Golgi complex from an epithelial cell of the male rat reproductive tract. EM: Golgi cisterna showing a concave central domain and an irregular peripheral domain • The Golgi complex is a stack of flattened cisternae. • It is divided into several functionally distinct compartments. • The collection of cisternae is broken down into cis, medial and trans compartments Two main network: a)cis Golgi network (CGN): face of the Golgi faces the ER (first cisternal structure) The cis Golgi network (CGN) functions to sort proteins for the ER or the next Golgi station. b) trans Golgi network (TGN) : the trans face is on the opposite side of the stack. The trans Golgi network functions in sorting proteins either to the membrane or various intracellular destinations. The Golgi Complex Regional differences in membrane composition across the Golgi stack: a. Reduced Osmium tetroxide in the cis cisternae b. Mannosidase II in the medial cisternae c. Nucleotide diphosphatase (split dinucleotides like UDP in the trans cisternae Fig 8.21  The Golgi complex is not uniform in composition; there are differences in composition from the cis to the trans face.  The Golgi complex plays a key role in the assembly of the carbohydrate components of glycoproteins and glycolipids.  It is the site of synthesis of complex polysaccharides, including proteoglycans and glycosaminoglycans chains, pectin and hemicellulose found in plants cell wall. © 2013 John Wiley & Sons, Inc. All rights reserved. The Golgi Complex The movement of materials through the Golgi complex It was generally accepted that Golgi cisternae were transient structures; however two contrasting views are:  In the vesicular transport model, cargo is shuttled from the CGN to the TGN in vesicles while the cisternae remains as stable element.  In the cisternal maturation model, each cistern “matures” as it moves from the cis face to the trans face.  Current model: similar to cisternal maturation model but with vesicle retrograde (backward) transport. Golgi cisternae serve an primary anterograde (forward) carriers. Fig 8.23 a,b The dynamics of transport through the Golgi complex © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Types of coated vesicles:  COPII-coated vesicles – move materials from the ER “forward” to the ERGIC and Golgi complex.  COPI-coated vesicles – move materials from ERGIC and Golgi “backward” to ER, or from the trans Golgi to the cis Golgi cisternae.  Clathrin-coated vesicles – move materials from the TGN to endosomes, lysosomes, and plant vacuoles. Proposed transport between membrane compartments of the biosyntheticsecretory pathway Fig 8.25a Fig 8.25b © 2013 John Wiley & Sons, Inc. All rights reserved. Summary Slide of vesicles transport and their functions Retrograde transport COPI COPII Anterograde transport Clathrin-coated vesicles Lysosomes Types of Vesicle Transport and Their Functions What determines the protein fate from ER to Golgi or its stay in ER? • A retrieval signal KDEL“lys‐asp‐gluleu” is on the soluble ER proteins. • Retrieval is accomplished as soluble ER proteins bind to KDEL receptors residing in the membranous wall of cis Golgi compartments. • The KDEL receptors, in turn, bind to proteins of the COPI coat, which allows the entire complex to be recycled back to the ER. Fig 8.28: Retrieving ER proteins Types of Vesicle Transport and Their Functions What determines the protein fate from ER to Golgi or its stay in ER? Proteins are maintained in a organelle by two mechanisms: a) Retention of proteins:  Retention of resident molecules that are excluded form transport vesicles is primarily based on the physical properties of the proteins.  Example: soluble proteins of ER lumen (chaperone that facilitate folding) or membrane proteins with short transmembrane domains are not likely to enter a transport vesicle. b) Retrieval of “escaped” molecules:  Retrieval of “escaped” molecules back to the compartment where they reside.  Resident proteins of the ER contains short amino acid sequences at their C terminus that serve as retrieval signals, ensuring their return to the ER if they accidently carried forward to the ERGIC or Golgi complex.   The retrieval signal is “lys‐asp‐glu-leu” (or KDEL in single‐letter nomenclature) These proteins are recognized and returned to the ER by the KDEL receptor  Each membrane compartment may have its own retrieval signals. © 2013 John Wiley & Sons, Inc. All rights reserved. Lysosomes  Lysosomes contain acid hydrolases which can digest every type of biological molecule.  A typical lysosome contains at least 50 different hydrolytic enzymes and produced in the RER. Portion of a phagocytic Kupffer cell of the liver showing at least 10 lysosomes of highly variable size © 2013 John Wiley & Sons, Inc. All rights reserved. Lysosomes: Autophagy Residual bodies become lipofuscin  Lysosomes play a key role in organelle turnover.  During autophagy, an organelle is surrounded by a double membrane and a structure called an autophagosome is produced.  The autophagosome is then fused with a lysosome to produce an autophagolysosome.  The digestive process leaves a residual body that can be secreted by the cell via exocytosis or become lipofuscin granules.  It is estimated that 1 to 1.5 % of the proteins Fig 8.32:A summary of the within a healthy liver cells are degraded via autophagic pathway autophagy per hour as part of a normal process of cellular renovation. © 2013 John Wiley & Sons, Inc. All rights reserved. The Human Perspective: Disorders Resulting from Defects in Lysosomal Function • • • • Lysosomal malfunctions can have serious effects on human health. Lysosomal storage disorders result from the absence of specific lysosomal enzymes thus allowing undigested material to accumulate. Lysosomal storage disorders can have a wide variety of symptoms. Among the best-studied disorders is Tay-Sachs disease. – It results from a deficiency in an enzyme responsible for degrading gangliosides, a major component of cell membranes. – It has a high incidence among Jews of eastern European ancestry. © 2013 John Wiley & Sons, Inc. All rights reserved. Autophagy Types: Phagophore is formed around the material to be digested Material fuses directly with the lysosome Chaperone mediated autophagy Selective degradation of mitochondria https://www.researchgate.net/figure/The-three-different-types-of-autophagy-a-macroautophagy-b-microautophagy-and-c_fig1_329793230 Exocytosis Exocytosis: discharge of a secretory vesicle or granule after fusion with plasma membrane.  Process is triggered by an increase in [Ca2+].  Contacts between vesicle and plasma membrane lead to formation “fusion pore”.  The luminal part of the vesicle membrane becomes the outer surface of the PM, and the cytosolic part becomes part of the inner surface of the PM. https://microbenotes.com/wp-content/uploads/2020/03/Exocytosis-Examples.jpg The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Endocytosis – uptake of cell surface receptors and bound extracellular ligands.  Endocytosis can divided into three categories:  Pinocytosis – nonspecific uptake of extracellular fluids.  Receptor-mediated endocytosis – uptake of specific extracellular ligands following their binding to receptors  Phagocytosis – uptake of particulate matter. https://www.nanolive.ch/cell-drinking-a-closer-look-on-macropinocytosis/ © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Endocytosis is a cellular process in which substances are brought into the cell. Endocytosis can divided into three categories: © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Phagocytosis Phagocytosis: The process of engulfment as illustrated by a polymorphonuclear leukocyte ingesting a yeast cell (lower left). Phagocytosis  For the uptake of large particles.  The plasma membrane takes up a particle and pinches off to form a phagosome.  The phagosome fuses with a lysosome and the material is digested within the phagolysosome. © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Phagocytosis Why all bacteria engulfed by phagocytosis cannot be destroyed? Hijacking Survival tools  The engulfment of particles by phagocytosis is driven by actin-containing microfilaments.  Not all bacteria engulfed by phagocytic cells are destroyed; some species hijack the phagocytic machinery for their own survival. – Mycobacterium tuberculosis (affects fusion with lysosome) – Coxiella burnetti (pH tolerant so can survive in the lysosome) – Listeria monocytogenes (can degrade the lysosome) Summary of the phagocytic pathway © 2013 John Wiley & Sons, Inc. All rights reserved.