Lesson 5 The ENDOMEMBRANE system 2020-21 PDF

CELL BIOLOGY UNIT 5 The Endomembrane System INTRODUCTION AND GENERAL CONCEPTS  Most eukaryotic cells have a complex array of internal membranes that form sacs, tubes and spheres distributed throughout the cytoplasm and create different intracellular compartments.  The presence of membrane-enclosed...

CELL BIOLOGY UNIT 5 The Endomembrane System INTRODUCTION AND GENERAL CONCEPTS  Most eukaryotic cells have a complex array of internal membranes that form sacs, tubes and spheres distributed throughout the cytoplasm and create different intracellular compartments.  The presence of membrane-enclosed compartments allows for segregation of processes or chemical reactions that could be mutually incompatible (for example some enzymes make peptide bonds while others hidrolyze them).  The resulting subdivision of the cytoplasm allows eukaryotic cells to function efficiently despite their large size (10.000 times the volume of a bacteria).  The endomembrane system is very dynamic and there is a continuous exchange of membrane within its different components and with the plasma membrane.  These internal membranes increase the area of cellular membrane enormously. The area of the endoplasmic reticulum alone is 20-30 times greater than that of the plasma membrane. INTRODUCTION AND GENERAL CONCEPTS The endomembrane system includes : Rough Endoplasmic Reticulum Smooth Endoplasmic Reticulum Golgi Apparatus Golgi Endosomes Lysosomes RER (only in animal cells) SER RER Lysosome ENDOPLASMIC RETICULUM The Endoplasmic Reticulum is a network of interconnected tubules and flattened sacs that enclose a single internal space called the ER lumen. The ER membrane is continuous with that of the nuclear envelope and represents about half the length of all cell membranes. The ER membrane is usually thinner than other cell membranes because it contains short chain fatty acids. Three domains can be distinguished within the ER :  The Rough Endoplasmic Reticulum (RER), which is covered by ribosomes (attached to RIBOPHORINS) on its cytoplasmic surface.  The Transitional Endoplasmic Reticulum, without ribosomes, where vesicles exit to the Golgi apparatus. Both the RER and the TER participate in protein processing.  The Smooth Endoplasmic Reticulum (SER), not associated with ribosomes and involved in lipid metabolism. RER SER ENDOPLASMIC RETICULUM FUNCTION The ER plays. Its membrane is the site of production of all transmembrane proteins and lipids for most organelles, including the ER itself, the Golgi apparatus, lysosomes, endosomes, vesicles and the plasma membrane. In addition secreted proteins will be synthesized in the RER.  The Rough Endoplasmic Reticulum (RER) : It is present in all cell types except in mature sperm cells and red blood cells (in mammals) but it is particularly abundant in secretory cells, pancreatic cells and hepatocytes. Its main functions are : Protein Synthesis (membrane or secreted) Protein Processing (glycosilation, proteolytic processing, folding …) Protein Sorting or Distribution.  The Smooth Endoplasmic Reticulum (SER) : It is present in all cells except in mammalian red blood cells. It is particularly abundant in striated muscle cells (sarcoplasmic reticulum), in Leydig cells in testis (steroid hormone synthesis) and in hepatocytes (lipid and lipoprotein synthesis). Its main functions are : Lipid metabolism Detoxification Intracellular Ca++ Storage THE RER AND THE SECRETORY PROTEIN PATHWAY George Palade´s Experiment (1960s) : Pulse-Chase experiment THE RER AND THE SECRETORY PROTEIN PATHWAY George Palade´s Experiment (1960s) : Pulse-Chase experiment Pancreatic acinar cells (secrete most digestive enzymes) were incubated with radioactive amino acids in order to radioactively label the newly synthesized proteins. The location of the labelled proteins was then revealed by autoradiography and electron microscopy. 1- Brief exposure to the radioactive amino acids labelled proteins are found in the RER 2- Brief exposure to the radioactive amino acids followed by a variable period without radioactive amino acids (chase period) the radiolabelled proteins were first detected in the Golgi apparatus, then in vesicles close to the plasma membrane and finally outside the cell. THE RER AND THE SECRETORY PROTEIN PATHWAY Palade’s experiments defined the pathway taken by secreted proteins : RER Golgi Secretory Vesicles Cell Exterior It was later established that proteins destined to the plasma membrane or to lysosomes follow the same route while proteins destined to the membrane or lumen of the ER or the Golgi apparatus only travel through the initial steps of the pathway. PROTEIN DESTINATION AND SITE OF SYNTHESIS Free Ribosomes Membrane-bound Ribosomes Golgi Retained in the ER Cytosol Free ribosomes and membrane-bound ribosomes are structurally identical. They only difer in the type of proteins they synthesize. SIGNAL SEQUENCES Signal sequences are short amino acid sequences at the N-terminal end of a protein that target either the nascent protein or the fully synthesized protein to a SPECIFIC CELLULAR LOCATION.  PROTEINS SYNTHESIZED ON FREE RIBOSOMES : If they don´t have a sequence signal they will stay in the cytosol. If they do have a target signal they will be sent to the destination determined by the specific signal : mitochondria, nucleus or peroxisomes.  PROTEINS SYNTHESIZED ON MEMBRANE-BOUND RIBOSOMES : They should have a specific signal sequence that will target the nascent protein during the translation process to the lumen of the RER. The fully synthesized protein will then stay in the RER or it will be sent to the Golgi apparatus and from there to lysosomes, to the plasma, nuclear or peroxisome membrane, or to the cell exterior. PROTEIN-SORTING IN EUKARYOTIC CELLS TRANSLOCATION OF PROTEINS TO THE RER  CO-TRANSLATIONAL TRANSLOCATION : Proteins are translocated to the rough endoplasmic reticulum during their synthesis on membrane-bound ribosomes. Most mammalian proteins enter the ER through this mechanism.  POST-TRANSLATIONAL TRANSLOCATION : Proteins are translocated to the RER once their synthesis has been completed on free cytosolic ribosomes. Yeast cells use both types of translocation. Once inside the ER, proteins will be subjected to further processing (folding, glycosylation, proteolytic cleavage...) COTRANSLATIONAL PATHWAY Step 1 : As the signal sequence of the nascent polypeptide (15-40 amino acids at the Nterminus) emerges from the ribosome it is recognized and bound by the Signal Recognition Particle (SRP). The Signal Recognition Particle (SRP) binds both the signal sequence and the ribosome and inhibits further translation. Step 2 : The SRP targets the entire complex (ribosome, mRNA and growing peptide chain) to the RER by binding to the SRP Receptor on the ER membrane. Signal Sequence SRP Step 3 : The SRP is released and the ribosome binds to a protein translocation complex or TRANSLOCON on the RER membrane (the SEC complex). The signal sequence is inserted into the membrane channel. SRP Receptor COTRANSLATIONAL PATHWAY Step 4 : Translation resumes and the growing polypeptide chain is translocated through the Translocon channel into the ER lumen. Ribophorins stabilize the binding of the ribosome to the membrane during this process. Step 5 : As translocation proceeds the signal peptide is cleaved by the Signal Peptidase and the polypeptide is released into the lumen of the ER. The ribosome dissociates and separates from the RER membrane. Transmembrane proteins destined to the plasma membrane or to other membranes are Signal Sequence not released into the ER lumen but are inserted into the ER membrane as the SRP polypeptide grows through the channel. SRP Receptor Signal Peptidase COTRANSLATIONAL PATHWAY : SUMMARY OF STEPS 1- Translation initiation and synthesis of the signal sequence on free cytosolic ribosomes. 2- The signal peptide is recognized by SRP (a ribonucleoprotein) and translation stops. 3- SRP binds to the SRP receptor on the RER membrane. 4- The ribosome associates to a translocator protein complex channel called SEC and SRP is released 5- Translation is re-initiated and the signal sequence is inserted into the channel. 6- The polypeptide is translocated through the channel into the ER lumen. 7- The peptide signal is cleaved by the signal peptidase and the protein is released into the ER lumen. POST-TRANSLATIONAL TRANSLOCATION PATHWAY Proteins are translocated to the RER after being fully translated on free cytosolic ribosomes.  These proteins also contain a signal sequence. They are stabilized in an unfolded conformation by cytosolic chaperones. Signal Sequence Cytosolic Chaperones  The peptide signal is recognized by a specific SEC complex associated to the translocation channel.  The SEC complex is also associated with a chaperone located on the inner side of the ER membrane : the BiP chaperone.  BiP pulls the protein through the channel and drives it into the ER lumen. ER Chaperone THE SMOOTH ENDOPLASMIC RETICULUM  It is a tubular network made of thin interconnected tubules whose membranes are a continuation of the RER membranes but without ribosomes.  Particularly abundant in cells with an active lipid metabolism such as adipose cells or hepatocytes , or cells that produce steroid hormones such as Leydig cells in testis and ovarian cells.  The SER tubules can establish connections with mitochondria, peroxisomes and glycogen granules. FUNCTIONS OF THE SMOOTH ER 1- It is the major site of membrane lipid synthesis in eukaryotic cells : Fatty acids and Phospholipids Cholesterol and its derivative hormones (steroids) Ceramide (precursor of glycosphingolipids and sphingomyelin) 2- Important for detoxification of various toxic lipid-soluble substances (in the liver) that are converted to water soluble compounds that can be eliminated through the urine. 3- Important for sequestering Ca2+ from the cytosol. The sarcoplasmic reticulum of the muscle cells is a specialized smooth endoplasmic reticulum that uptakes Ca2+ from the cytosol using a Ca2+-ATPase pump. The release of Ca2+ from the SR and its reuptake trigger the contraction and relaxation of myofibrils during each round of muscle contraction. BIOSYNTHESIS OF MEMBRANE LIPIDS Because they are extremely hydrophobic, lipids are synthesized in association with already existing cellular membranes rather than in the aqueous environment of the cytosol. The majority are synthesized in the SER. Once synthesized they are transported from the ER to their ultimate destination either in vesicles or with the help of carrier proteins. Most phospholipids (major components of biological membranes) are derived from GLYCEROL. Fatty acids are linked to Coenzyme-A carriers to form Acyl-CoAs and the polar head molecules (choline, ethanolamine, serine, inositol) are transferred linked to a dinucleotide phosphate (CDP) as CDP-choline, CDP-ethanolamine … BIOSYNTHESIS OF MEMBRANE LIPIDS New Phospholipids  New phospholipids are synthesized and inserted on the cytosolic side of the SER membrane.  To maintain a stable membrane some of the newly synthesized phospholipids must be transferred to the other half of the bilayer.  This transfer process IS NOT SPONTANEOUS and requires the enzymatic activity of FLIPPASES. These membrane proteins catalyze the rapid translocation of phospholipids across the SER membrane. Cytosol SER Lumen FLIPPASE Cytosol SER Lumen New Phospholipids DETOXIFICATION PROCESS Toxic substances such as lipid-soluble drugs or various harmful compounds produced by metabolism, must be eliminated from the organism. These substances are produced upon degradation of foreign cells or come from drugs, herbicides, preservatives …. Detoxification occurs mostly in the SER of liver, kidney, intestine, lung and skin cells. The detoxification process involves oxydations mediated by non-specific oxidorreductases like Cytochrome P450, and further processing to transform lipid-soluble toxic compounds into water-soluble substances. These soluble molecules are then transferred to the bloodstream and eliminated through the urine. THE GOLGI APPARATUS It was discovered by the italian scientist Camillo Golgi in 1898. It is composed of :  A stack of discrete (not connected) membrane-enclosed sacs or cisternae (4 to 6). Each stack is a DICTIOSOME and together, all cellular dictiosomes form the Golgi apparatus.  A collection of transition vesicles coming from the ER and budding vesicles exiting from the Golgi stacks towards the plasma membrane or other cellular compartments. STRUCTURE OF A DICTYOSOME A dictiosome shows FUNCTIONAL AND STRUCTURAL POLARITY : 1- The Cis face or formation face: the one closest to the ER and the nucleus (proximal). It is the entry face for vesicles coming from the ER. It is the convex side of the stack. 2- The Medial region: the middle region where molecular modifications take place. 3- The Trans face or maturation face : the one closest to the plasma membrane (distal). It is the exit face for secretion vesicles or vesicles leaving for other locations (plasma membrane, lisosomes). It is the concave side of the stack. FUNCTIONS OF THE GOLGI APPARATUS  POST-TRANSLATIONAL MODIFICATION of proteins synthesized in the ER :  GLYCOSYLATION : N-glycosylation (partially) and O-Glycosylation.  PROTEOLYSIS : specific proteolytic cleavage of certain protein regions  SULFATION : addition of sulfate groups to certain proteins.  SORTING AND PACKING of proteins on specific vesicles depending on their final destination.  A different modification occurs in each sac or cisterna  Proteins move from one cisterna to the next packed into vesicles  Proteins do not need to go through all the cisternae THE DYNAMICS OF THE GOLGI APPARATUS The Golgi Apparatus is part of a dynamic system of intracellular trafficking that transfers biomolecules from one part of the cell to another  Proteins travel from the ER to the Golgi apparatus in vesicles. The enzymes present in the Golgi sacs detect specific sequence motifs that will determine how the proteins will be modified. This modifications in turn will define the ultimate cellular destination of these proteins : lysosomes, plasma membrane or secretion.  Glycosphingolipids and Sphingomyelin are also synthesized in the Golgi apparatus from their precursor Ceramide (whose synthesis occurs in the SER). POST-TRANSLATIONAL PROCESSING Post-translational processing is required for proteins to acquire their mature functional conformation and to be sent to their final destination. It includes :  Proteolysis (specific proteolytic cleavage)      Folding Disulfide bond formation Glycosylation Phosphorylation Addition of lipids (lipidation) PROTEOLYTIC CLEAVAGE Specific cleavage of the polypeptyde chain is an important step in the maduration process of many proteins. 1) Removal of the initiator methionine from the amino terminus of many proteins. 2) Removal of the signal sequence: the amino-terminal signal peptide is cleaved as the polypeptide is translocated through the ER membrane. This type of cleavage is essential for translocation of proteins across membranes and it allows other chemical groups to be added to the N-terminus of some proteins. 3) Cleavage of larger protein precursors to obtain active enzymes or hormones. For example, Insulin is synthesized as preproinsulin and removal of the signal sequence yields proinsulin that will be converted to insulin by proteolytic removal of an internal peptide. PROTEIN FOLDING MOLECULAR CHAPERONES are proteins that facilitate the folding of other proteins into their native three-dimensional conformation.  HSP70 Chaperones : they bind and stabilize unfolded or partially folded polypeptides during translation or during their transport to different cellular locations. Chaperone binding prevents aggregation or degradation of unfolded polypeptides. They are present in the cytosol, ER (BiP), mitocondria and chloroplasts. Chaperone  HSP60 Chaperonins : they directly facilitate folding of proteins into their final structure.They are present in the cytosol, mitocondria and chloroplasts. In the cytosol Hsp70 and chaperonins act sequentially. They require ATP to release the protein. Hsp60 Hsp70 https://www.youtube.com/watch?v=--NcNeLc1mo PROTEIN FOLDING Two enzymes also contribute to protein folding 1) Protein Disulfide Isomerase (PDI) : formation of disulfide bonds Stabilization of the folded structure of some proteins often require the formation of disulfide bonds between Cysteine residues. In eukaryotic cells, disulfide bonds are formed in the rough endoplasmic reticulum and are generally restricted to secreted proteins and some membrane proteins. 2) Peptidyl Prolyl Isomerase catalyzes the interconversion of cis-Proline into transProline and, as a result, the isomerization of peptide bonds that involve Proline. This isomerization stabilizes the beta-turns that include Proline. PROTEIN GLYCOSYLATION Glycosylation is the addition of branched oligosaccharides to proteins. These proteins, called glycoproteins are usually secreted or localized to the cell surface. Glycosylation occurs in the ER and in the Golgi apparatus. There are two types of carbohydrate modifications:  N-GLYCOSYLATION : binding of sugar residues to an amino group in the lateral chain of Asparagine (Asn) through an N-glycosidic bond. It starts in the ER and continues in the Golgi apparatus.  O-GLYCOSYLATION : binding of sugar residues to a hydroxyl group (-OH) in the lateral chain of Threonine through an O-glycosidic bond. Only in Golgi. N-GLYCOSYLATION  It starts in the ER lumen and continues in the Golgi apparatus  The oligosaccharide is synthesized on a lipid carrier anchored in the ER membrane : DOLICHOL. It is then transferred as a unit to the amino group in Asparagine.  The oligosaccharide initially contains N-acetylglucosamine (2), Mannose (9)and Glucose (3).  Subsequent trimming eliminates 3 Glucose residues and one Mannose.  Modification of the oligosaccharide will continue in the Golgi cisternae where a variety of different N-linked oligosaccharides are formed. ER Lumen DOLICHOL Cytosol O-GLYCOSYLATION  It takes place exclusively in the Golgi apparatus  Sugar residues are linked to the OH group of Threonine or Serine  Sugar molecules are added sequentially (one by one) with the help of Glycosyltransferases.  Monosacharides are transferred bound to UDP or to CMP (nucleotides diphosphate or monophosphate)  Sugar chains are shorter than in N-glycosylation NAcGal Gal NAcGal : N-acetylgalactosamine NANA : N-acetylneuraminic acid (sialic acid) NANA Differences between N-glycosylation and O-glycosylation Asparagine N-Glycosidic bond N-acetylglucosamine Addition in block Longer oligosaccharide Threonine / Serine O-Glycosidic bond N-acetylgalactosamine Addition one by one Shorter oligosaccharide IMPORTANCE OF GLYCOSYLATION 1) Glycosylation promotes protein folding in the ER 2) Glycoproteins are more resistant to digestion by proteases. 3) Acts as a protective coat in plasma membrane 4) Important for cell-cell recognition processes. 5) Regulatory roles and signaling. OTHER PROTEIN MODIFICATIONS  SULFATION : addition of sulfate groups (SO42- ). Typical of proteoglycans (ChondroitIn sulfate, dermatan sulfate, heparan sulfate and keratan sulfate) secreted mostly by condroblasts and osteoblasts. Takes place in the Golgi apparatus.  PHOSPHORYLATION : addition of phosphate groups. Catalyzed by PROTEIN KINASES. These enzymes transfer a phosphate group from ATP to hydroxyl groups on the lateral chain of Serine, Threonine or Tyrosine. Occurs mostly in the nucleus and the cytosol (but not exclusively) Phosphorylation can be reverted by PROTEIN PHOSPHATASES that catalyze the removal of phosphate groups from phosphorylated proteins. Kinase Phosphatase CELL COMPARTMENT POST-TRANSLATIONAL MODIFICATION Cytosol Folding Proteolysis Phosphorylation RER Golgi N-glycosylation (ends) O-glycosylation Sulfation Nucleus Phosphorylation Mitochondria Folding Proteolytic Cleavage Folding Proteolytic Cleavage N-Glycosylation (start) Disulfide bond formation LYSOSOMES and ENDOSOMES ENDOSOMES An endosome is a vesicle formed by the invagination and pinching off of the cell membrane during endocytosis. Fate of endosomes: 1- Fusion with lysosomes :the endocytosed material will be degraded. 2- Recycling back to the membrane :Part of the endocytosed material will be recycled back to the membrane (cell surface receptors devoid of their ligand) 3- Transcytosis : transport through the cell from one side to the other. LYSOSOMES LYSOSOMES are the Principal Sites of Intracellular Digestion of Macromolecules  They are membrane-enclosed organelles that contain an array of digestive enzymes (nucleases, proteases, lipases, glycosidases...) capable of digesting all biopolymers.  Lysosomes look like dense, spheric vacuoles of different size and shape.  All lysosomal enzymes are maximally active at acidic pH : they are ACID HYDROLASES.  Low pH is maintained by a H+ pump on the lysosomal membrane, an active transporter that requires ATP hydrolysis to “push” cytosolic H+ inside the lysosome. LYSOSOME FORMATION OR MATURATION Mature lysosomes (secondary lysosomes) arise from the fusion of : endocytosis Transport vesicles budded from de Trans Golgi Network containing acid hydrolases. Endosome Endosomes with endocytosed molecules from the cell surface. The formation of mature lysosomes represents an intersection between the secretory pathway and the endocytic pathway. Vesicles with acid hydrolases Golgi Lysosome SORTING AND PACKAGING OF PROTEINS VESICULAR TRANSPORT  Newly synthesized proteins are sent to the cis Golgi on vesicles generated in the transitional ER.  The proteins will leave the Golgi apparatus on vesicles that bud from the trans face. Some of these proteins will be anchored into the membrane and will be transported as vesicle membrane proteins to their final destination (plasma membrane, nuclear membrane, peroxisome membrane...). Others will be soluble proteins that will be secreted (or sent to lysosomes).  Vesicle transport depends on motor proteins associated to Microtubules and on specific receptors or adaptor proteins on the vesicle surface.  Transported vesicles can fuse with the plasma membrane or with other organelles membrane. The final destination of vesicles is different depending on their protein content: 1. Constitutive secretion vesicles 2. Stimulus-regulated secretion vesicles 3. Vesicles that travel to different cell organelles depending on their content (lysosomes, peroxisomes...) Vesicle-Coating Proteins Vesicles coated with different proteins are involved in transport between different cell compartments or in different transport pathways.  COPII (coat protein II) decorates vesicles that transport their content from the endoplasmic reticulum (ER) to the Golgi (cis face).  COPI (coat protein I) decorates transport vesicles that move in the opposite direction, from the Golgi to the ER (retrograde transport). COPI also forms vesicles for intra-Golgi transport.  CLATHRIN – coated vesicles are involved in transport : from the trans-Golgi network to endosomes. from endosomes to lysosomes or to the plasma membrane. from the plasma membrane to early endosomes (endocytosis). 48