ER Structure and Function PDF

MED100-I Medical Biology Structure and Function of the Endoplasmic Reticulum Molecular mechanisms of protein synthesis in Rough Endoplasmic Reticulum  Endoplasmic Reticulum - endoplasmic - “within the cytoplasm” - reticulum - Latin for a “a little net” - exten...

MED100-I Medical Biology Structure and Function of the Endoplasmic Reticulum Molecular mechanisms of protein synthesis in Rough Endoplasmic Reticulum  Endoplasmic Reticulum - endoplasmic - “within the cytoplasm” - reticulum - Latin for a “a little net” - extensive network of folded membranes that extends from the nuclear envelope to which it is connected, throughout the cytoplasm  Endoplasmic Reticulum a netlike labyrinth of branching tubules and sacs that extends from nucleus to plasma membrane Sacs (cisternae), internal space, ER lumen Tubules The sacs and tubules are all interconnected by a single continuous membrane  Endoplasmic Reticulum Intracellular channels The endoplasmic reticulum serves many general functions, including the folding of protein molecules in sacs called cisternae and Intracellular transport the transport of synthesized proteins in vesicles to the Golgi Lipid biosynthesis apparatus Protein biosynthesis  There are two basic kinds of endoplasmic reticulum 1- Rough (granular ) ER 2- Smooth (agranular) ER (RER or GER) (SER) The surface of the RER is studded with ribosomes giving it a "rough" appearance The quantity of RER and SER in a cell can interchange from one type to the other, depending on changing metabolic needs.  Microsomes RER and SER regions of ER can be seperated by centrifugation. When tissue distrupted by homogenization the ER breaks into many small (100-200 nm in diameter) closed vesicles called microsomes. Microsomes are useful for studying of ER functions in vitro.  Smooth ER (SER): Regions of ER that lack bound ribosomes are called smooth endoplasmic reticulum, *Tubular or vesicular in form *SER membranes arise from GER *SER is not involved in protein synthesis *In the different cells the function of SER are different The SER functions SER has different functions in the specialized cells 1- biosynthesis of lipids 2- lipid transport 3- biosynthesis of steroid hormones 4- the metabolic reactions in the liver cells 5- contraction process in the muscle cells 6- regulation in neuronal synapse 1- SER is involved biosynthesis of lipids The synthesis of fatty acids and phospholipids occurs in the smooth ER The ER also produces cholesterol SER is the principle site of production of lipoprotein particle in liver The enzymes that synthesize the lipid component of lipids are located in the SER membrane 2- SER functions in lipid transport Dietary lipids breakdowns into fa and mg by pancreatic lipase in the small intestine absorptive epithelial cells; monoglycerides , fatty acids absorbtion-pinocytosis intestine cell SER (triglycerides) Acyl-CoA synthetase, acyltransferases Golgi Complex (apolipoproteins chylomicron )) lymphatic or blood vessels fats are mainly digested in the small intestine by pancreatic lipase the bile salts emulsifies them. That is, they break the big droplet into many smaller ones. Helps to absorbtion Complete digestion of fat (a triglyceride) results in fatty acid and monoglycerol molecules the mechanism of lipid absorption: - emulsification, - lipolysis, - micellar formation, - membrane translocation, - intracellular resynthesis, - chylomicron formation, - lymphatic drainage. 3- SER functions in biosynthesis of steroid hormones: It contains some of the enzymes required for steroid synthesis and they are abundant in ; *Leydig’s cells of testes: testesteron *Adrenal cortex cells: corticosteroids *Corpus luteum cells of ovarium: progesteron *Leydig’s cells adrenal cortex  4- SER is involved in the metabolic reactions in the liver cells (SER is abundant in hepatocytes) A- SER contains specific enzymes to detoxify (to break down) drugs, alcohol, steroid hormones and toxic chemicals; cytochrome P450 enzymes It is also called CYP enzymes The CYP enzymes catalyze the oxidation of organic substances. CYPs are the major enzymes involved in drug metabolism. Smooth ER plays a large part in detoxifying a number of organic chemicals converting them to safer water-soluble products. Human CYPs Humans have 57 genes cytochrome P450 genes 4- SER is involved in the metabolic reactions in the liver cells B- it is involved in the breakdown of glycogen into glucose Smooth ER also contains the enzyme glucose-6-phosphatase which converts glucose-6-phosphate to glucose, (a step in gluconeogenesis) The liver is also the main site in the body for gluconeogenesis Glycogen SER  5- SER participates in the contraction process in muscle cells A special type of smooth ER is found in smooth and striated muscle called “Sarcoplasmic reticulum;SR” The SR consists of a branching network of SER cisternae surrounding each myofibril  The sarcoplasmic reticulum stores and pumps calcium ions, specifically regulates Ca+2 flow in muscle cells SER membrane contains Ca+2 - ATPase pumps Ca+2 regulate muscular contraction The SR's release of Ca+2 upon electrical stimulation of the cell plays a major role in excitation-contraction coupling when the muscle is exitated, Ca+2 diffuse out of the SR in the resting state, most of the Ca+2 reside in the SR 6- Regulates neuronal synapse “a synapse is a structure that permits a neuron to pass an electrical or chemical signal to another cell.” In neuron, the end of the axon (synaptic terminal) contain a number of SER vesicles. during synapse SER is involved to Ca+2 regulation similar to myocytes.  GRANULAR ER (GER) Rough ER (RER) http://upload.wikimedia.org/wikipedia/commons/thumb/f/f6/Nucleus_ER_golgi.svg/365px-Nucleus_ER_golgi.svg.png  GER is involved in protein synthesis GER prominent in cells for protein secretion. In light microscope, it can be detected by staining with basic dyes (e.g. Nissl bodies in motor neurons) GER Nissl bodies: GER+ Ribosome clusters rer1 In electron microscope, GER appears as parallel membrane limited flattened sacs or cisternae The membranes of the ER are continuous with the outer https://figures.boundless.com/2718/full/422px-nucleus-er.svg.png membrane of the nuclear envelope. http://pattonscellorganelles.weebly.com/uploads/8/7/2/4/8724916/5693747.jpg?406  GER is particularly well developed in protein secreting cells in the digestive enzyme producing cells of the exocrine pancreas; aciner cells in the collogen and elastin producing cells of the connective tissue; fibroblast in the antibodies producing cells; plasma cell in the neurotransmitter producing cells; motor neurons in the phagocytic cells containing lysosomal enzymes ; macrophages Liver cell Plasma cell Aciner cell the rough ER in a pancreatic exocrine cell that The hepatocyte is a cell in the body that manufactures makes and secretes large amounts of digestive serum albumin, fibrinogen, and the prothrombin group of enzymes every day. The cytosol is filled with clotting factors closely packed sheets of ER membrane studded with ribosomes The GER has a central role in protein biosynthesis and their transportation within the cell  How do the right proteins get to the right places? The mechanism of the protein synthesis in GER is explained with SIGNAL HYPOTHESIS The signal hypothesis, formulated by Günter Blobel and David Sabatini in 1971, and elaborated by Blobel and his colleagues between 1975 and 1980 SIGNAL HYPOTHESIS Günter Blobel, (Nobel Prize for Medicine - 1999) 1999 Nobel Prize in Physiology has been awarded to Günter Blobel (the Rockefeller University,) for ''the discovery that proteins have intrinsic signals that govern their transport and localization in the cell''.  SIGNAL HYPOTHESIS Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific subcellular location or exported from the cell for correct activity. This phenomenon is called protein targeting (signal hypothesis) Protein targeting is necessary for proteins that are destined to work outside the cytoplasm This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases. Sorting or translocation can occur as 1. CO TRANSLATIONAL TRANSLOCATION Synthesized protein is transferred to an SRP receptor on the endoplasmic reticulum (ER). There, the nascent protein is inserted into the translocation complex 2. POSTTRANSLATIONAL TRANSLOCATION Even though most proteins are co translationally translocated, some are translated in the cytosol and later transported to their destination. This occurs for proteins that go to a mitochondrion, a chloroplast, or a peroxisome co-translational targeting (secretory pathway): ER Golgi lysosomes plasma membrane secreted proteins post-translational targeting: nucleus mitochondria Peroxisomes  According to this hypothesis; Three different types of the protein that are the related to the GER are synthesized in cells. 1- Proteins that are secreted from the cell 2- Lysosomal enzymes 3- Plasma membrane glycoproteins  In cells, molecular labels (often, amino acid sequences) are used to "address" proteins for delivery to specific locations. A characteristic feature of these targeting pathways (with the exception of cytosolic and nuclear proteins) is the presence of a short amino acid sequence at the amino terminus of a newly synthesized polypeptide called the signal sequence or signal peptide. In 1975, George Palade, at the Rockfeller Institute in New York, demonstrated that proteins with these signal sequences are synthesized on ribosomes attached to the ER membrane. Targeting signals are the pieces of information that enable the cellular transport machinery to correctly position a protein inside or outside the cell. In the absence of targeting signals, a protein will remain in the cytoplasm. ❑ If an mRNA lacks a signal sequence protein will be synthesized entirely in the cytoplasm on free ribosomes as a structural protein of the cell ❑ If an mRNA contains “signal sequence” initially mRNA binds to the free ribosomes in the cytoplasm, the signal peptide is synthesized on the ribosome in the cytoplasm.  The signal peptide is about 16-20 amino acids and it appears at the beginning of the polypeptide chain. As the signal peptide emerges from the ribosome it is recognized by a special molecule in the cytosol (signal recognation particles; SRP) (SRP;6 proteins +7S RNA molecule) takes the ribosome to the ER.  SRP binds to the signal peptide and ribosome SRP binding sites; 1- signal peptide then “SRP and ribosome complex” attaches to the 2- ribosomal A site ER at specific sites called SRP receptor 3- SRP receptor on ER For transport of a polypeptide into the ER lumen, the signal sequence attaches to the SRP receptor. The hydrophobicity of the signal sequence is postulated to be the molecular key for the polypeptide's interaction with the ER membrane, which is also a hydrophobic structure. The second recognition site, ribosome receptor, serves to anchor the organelle (ribosome) to the ER membrane. SRP signal peptid The interaction between the signal sequence and the ER membrane is believed to open a channel in the membrane through which the polypeptide is transported into the ER lumen. Thus, the molecular instructions for transport into the ER (in the form of a hydrophobic sequence) are furnished by the polypeptide. SRP receptor ER lumen  SRP binds to the signal peptide and ribosome (A site) (At this moment protein synthesis is arrested) SRP and ribosome complex attaches to the ER at the specific sites called SRP receptor (or docking protein)  Upon binding to the docking SRP receptor protein, SRP is released from ribosome, GER lumen returns to cytosol it may participate in other SRP round of protein synthesis cycle Signal peptide ribosome  on the ER membrane there are pore proteins (Translocator proteins; translocon ), ribosomal large subunits attaches to this pore proteins. translocon; Sec61p complex When SRP is released from ribosome, at this moment protein synthesis starts, The signal peptide and the growing polypeptide chain is released through the translocon into the cisterna (lumen) of GER  The signal peptide is cleaved from the polypeptide chain by signal peptidase into the ER lumen. The last step; when the protein synthesis is complated the ribosomes detaches from the GER membrane, then mRNA and ribosomes may participate in an other round of protein synthesis.  Original figures The signal hypothesis as it was proposed by Günter Blobel and David Sabatini in 1971  Post-Translational Modifications in the Rough ER Newly synthesized polypeptides in the membrane and lumen of the ER undergo principal modifications before they reach their final destinations: 1. Specific proteolytic cleavages 2. Addition and processing of carbohydrates 3. Proper folding 4. Assembly into multimeric proteins 1- Specific proteolytic cleavages The signal peptide is removed from the polypeptide by the SIGNAL PEPTIDASE (protease) 2- Addition and processing of carbohydrates GLYCOSYLATION Many secreted and membrane proteins contain covalently attached carbohydrates (CH) Addition of CH chains starts in ER , terminates in Golgi Complex  Precursor oligosaccarides (OlSc )are synthesized in the cytosol, then imported into the ER or Golgi lumen by Dolichol (special lipid molecule) which holds OlSc chains in ER membrane (flip-flop )  CH (oligosaccarides) The precursor ofchains arechains the CH boundattached to specific to site the on ER the polypeptide; membrane by a dolichol lipid carrier (phospholipid) They are“Asparagine-X transferred to-serin/threonin” the polypeptide. This process is catalyzed by various oligosaccharyl transferases 3- Proper folding: translocated polypeptide chains fold and assembled in ER lumen Each polypeptide has a different folding pathway, dictated by its sequence, The folding of many newly made proteins within the GER is facilitated by some folding catalysts; 1- BIP; Binding protein assists protein folding the Sec61 channel, and another chaperon within the ER called BiP is required to pull the polypeptide chain through the channel and into the ER 2-Folding proteins; Calnexin and Calreticulin (lectins) 3- Disulfide bonds; disulfide isomerase  Formation of disulfide bonds (-s-s-) Disulfide bonds (DBs) help stabilize the tertiary and quaternary structure of many proteins (e.g. insulin) In eukaryotic cells, DBs are formed in the GER, but not in the cytosol Protein disulfide isomerase ,an enzyme localized to GER lumen, catalyzes the rearragement of disulfide bonds. DBs are common in secretory proteins and exoplasmic domains of membrane proteins, but are absent from soluble cytosolic proteins  Only properly folded proteins are transported Proteasome pathway from the GER to the Golgi Abnormally folded or unfolded proteins are exported from ER then they are degraded in the ubiquitin- proteasome pathway in the cytosol Otherwise improperly folded proteins are retained in the GER and result in ER stress,  Proteins can be translocated into the ER either during their synthesis on membrane-bound ribosomes (cotranslational translocation) or after their translations has been completed on free ribosomes in the cytosol (posttranslational translocation) Soluble proteins Plasma membrane glycoproteins  Soluble proteins destined for the ER lumen, for secretion, or for transfer to the lumen of other organelles pass completely into the ER lumen. rer15  Plasma membrane glycoproteins synthesis Transmembrane proteins destined for the ER or for other cell membranes are translocated to the ER membrane and remain anchored there by one or more membrane-spanning a-helical regions in their polypeptide chains. Co-translational protein insertion In mammalian cells, most proteins enter the ER co- translationally Plasma membrane glycoproteins synthesis Co-translational protein insertion Topologies of the integral membrane proteins synthesized on the rough ER  Plasma membrane glycoproteins synthesis Post-translational protein insertion rer20 It was discovered that many proteins can enter ER membranes posttranslationally, that is after much or all of their synthesis is complete. ATP is required for this process The hydrophobic portions (signal pepdides) of the protein can act either as start-transfer or stop-transfer signals during the translocation process. rer21 When a polypeptide contains multiple, alternating start-transfer and stop- transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein.  Export of proteins from the ER to Golgi complex for the further modifications * newly synthesized proteins travel along the secretory pathway in transport vesicles, which bud from the membrane of one organelle (ER) and then fuse with the another organelle membrane (Golgi).  Many resident ER proteins participated in the ER functions (e.g BIP) can escape from ER. they are returned to the ER from the Golgi apparatus The ER resident proteins contain short aa sequences (signal) called the KDEL sequences gol2  The Golgi complex has KDEL receptors Golgi traps the ER proteins and selectively transports back to the ER.  ER Stress Various conditions can disturb ER functions, including inhibition of protein glycosylation, reduction of formation of disulfide bonds, calcium depletion from the ER lumen, impairment of protein transport from the ER to the Golgi, expression of malfolded proteins. Such ER dysfunction causes proteotoxicity in the ER, termed "ER stress” In mammalian cells, ER stress occurs under many conditions; pharmacological chemicals, decreases in oxygen, glucose, ATP and calcium ions, nutrient deprivation, developmental processes, genetic mutations, pathogenic insult. In order to survive under ER stress conditions, cells have a self-protective mechanism against ER stress, the ER stress response ; unfolded protein response, UPR Numerous pathophysiological conditions are associated with ER stress-induced apoptosis including ischemia, diabetes and neurodegenerative diseases.  There are four functionally distinct unfolded protein response, UPR The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of ER. 1-Transcriptional induction of ER chaperones increases protein folding activity and prevents http://www.naika.or.jp/im2/42/01/figs/03/fig1.jpg protein aggregation 2- Translational attenuation reduces the load af new protein synthesis and prevent further accumulation of unfolded proteins 3- The ER associated degradation (ERAD) pathway eliminates misfolded proteins by the ubiquitin-proteasome system 4- If the stress cannot be resolved, severe and prolonged ER stress extensively impairs the ER functions\ then the cell dies by apoptosis.