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The endoplasmic reticulum.docx

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BIOL 2020

The endoplasmic reticulum (ER) When we mash up a cell- majority of microsomes come from the ER Major amount of membrane in a cell in the ER (jammed packed with membrane) Two categories Rough (RER): Ribosomes bound to cytosolic surface of ER Some ribosomes not attached = free ribosomes Smooth (SER): no ribosomes, connected to the RER RER and SER: many other differences January 27th (Lecture 9) Endoplasmic reticulum (ER) Protein reticulon (group of them) when they isolate the ER (by mashing the cell) these critters (organelles) form vesicles called microsomes If you isolate the smooth ones you can ring a lot of this protein (reticulon) The RER doesn't have nearly as much Ribosomes inside the cisternae are called free ribosomes RER vs SER RER: Extensive membrane Interconnected sacs - cisternae Continuous with nuclear envelope SER: More tubular Interconnecting pipelines Continuous with RER (SEE SLIDE 7 - CELL 4 ER) The inside of the rough ER is called the lumen (also referred to the inside of organelles) Means inside Cisternal space = lumen Scanning electron microscope: coat the 3D shape with electron dense metals and then we bombard it with electrons and measure the elections that bounce off SER functions Synthesis of lipids Majority of lipids are synthesized here Phospholipids and sphingolipids are synthesized in the P face and then flippase flips them to the E face Skeletal muscle In skeletal muscles, they play a major role in storing calcium - sarcoplasmic reticulum (synonymous with SER) Muscle contractions need calcium Steroid producing cells Steroid: 4 ring structure that makes up sex hormones Liver detoxification Especially evident in the liver SER is crucial to detoxify molecules A lot of things that we eat and drink are hydrophobic and we need to make them hydrophilic Enzyme cytochrome p40 (take O2 and add OH - will now make the molecule more water soluble and we can metabolize it etc) RER functions (protein synthesis) Polypeptides synthesized at two locales ONE: On ribosomes attached to cytosolic surface of RER Proteins secreted from the cell Integral membrane proteins Soluble proteins within ER, golgi, lysosomes, endosomes TWO: Other polypeptides - on free ribosomes Released into the cytosol Enzymes Peripheral proteins - plasma membranes Proteins - nucleus, mitochondria, peroxisomes Translation (KNOW THIS WORD): protein synthesis through a ribosome Can make a protein on ribosomes attached to the RER, which produces a protein that is different than the one made on the free ribosomes Cytosolic ribosomes = free ribosomes Peripheral proteins on the E face need to be secreted and then bound - we are talking about the proteins on the P face How are proteins synthesized at different sites? (SEE SLIDE 12 - CELL 4 ER) Small binds to mRNA and then bring in the large tRNA decodes the message then forms a bond…. Ribosomes SEE SLIDE 14 (CELL 4 ER) Ribosomes are assembled int he nucleus in the nucleolus Different types of RNA mRNA: codes for proteins tRNA: used in protein synthesis (carries amino acids) rRNA: forms part of the structure of the ribosome and essential for ribosome function Small RNAs: involved in pre-mRNA splicing (SEE SLIDE 16 - CELL 4 ER) 60s + 40s - get 80s because the shape plays a big role in the sedimentation We make 70S ribosomes in the mitochondria Mitochondria used to be bacteria Ribosomal RNA All have roughly the same structure Reason why it doesn't matter whether you're a bacteria or a human is because you make proteins RNA can make a duplex single strand RNA can base pair with RNA RNA can be a catalytic enzyme What does ribosomal RNA code for? Nothing - it doesn't code - not an information molecule in coding - only mRNA codes (for proteins) A: Amino acyl cite (tRNA comes in) P: Peptide bond E: Exit site (SEE SLIDE 20 - CELL 4 ER) 2 subunits (small and large) There is a place on the ribosome where the amino acid comes in - first amino acid is methylamine for EVERY protein mRNA binds to the small subunit When the protein is being made its not exposed because all of the early synthesis occurs where the amino acids are buried in a tunnel (can hold about 30 amino acids) Tunnel is on the large subunit January 29th (Lecture 10) Small subunit sometimes called embryo Embryo held by the big subunit (SEE SLIDE 25 - CELL 4 ER) Summary slide for some things that go on Transcription makes mRNA Translation only occurs in the cytosol/cytoplasm In the nucleus we have 1000s of proteins All of those were made in the cytoplasm and had to go back into the nucleus (got transported back) rRNA made in nucleolus - proteins start assembling the ribosomes in the nucleolus Ribosomes partially assembled in the nucleolus Secretory pathway Transport-secretion is a complex process Don't know what the vesicles are doing at that exact moment (in picture) A lot of stuff going on Pulse-chase (George Palade) SEE SLIDE 3 (CELL 5 SECRETION) Done in the 60s What is the pathway of secretion in a cell? Where do things get made and how do they move through the cell and eventually get secreted? Make pathway radioactive - use radioisotopes Chose to make the proteins radioactive Radioisotope: molecule that has a different amount of neutrons (isotope) Carbon-12 (stable) has 6 protons and 6 neutrons Can also exist as carbon-13 (stable) Carbon-14 (unstable - gives off radiation - energy) All 3 of these our body cannot recognize as being different Works, acts and looks identical Fed cells with radioactive amino acid - can choose any one you want (most often methylamine) Detecting radioactivity can be done with X-ray film or liquid In first 3 minutes - radioactive protein was found on the RER Looking for electron dense regions not found in normal cells Saw that there were membranes with black dots (which turned out to be ribosomes) Radioactivity was then in the golgi - no longer in the RER After another 120 minutes - coated cells with emulsion - now radioactivity was in vesicles and being secreted out of the cell Secretory pathway RER --> Golgi --> Vesicles --> Outside Secretion occurs only on 1 face of the membrane Secretory proteins are synthesized in the ER and pass through the golgi on the way to the extracellular environment SEE SLIDE 5 (CELL 5 SECRETION) Pancreatic cells Pancreas: makes hormones and digestion (makes enzymes that chew up our food) That food has to go to the apical membrane – where the cell undergoes secretion The rest of the membrane has different names Rest of the membrane is called basal lateral membrane Want secretion to go to a duct Secretion only occurs in one area of the cell – apical Killer cells: recognize tumor cells and virally infected cells – swimming around in our blood – see a tumor cell and bind to it – this cell doesn’t initially have an apical membrane but when it binds the golgi moves and the vesicles moves towards the tumor cell – bind to tumor and inside those vesicles will release molecules (some put holes in the cell) also release enzymes that trigger apoptosis We don’t kill innocent bystanders – of normal cell is close to tumor cell and killer cell – the normal cell won’t die Five steps in the maturation of secretory proteins as defined by yeast Sec mutants temperature sensitive for protein secretion SEE SLIDE 6 (CELL 5 SECRETION) Randy Shekman Works with yeast - - mutated yeast and selected yeast that had mutation in the secretary pathway Temperature sensitive mutants at low temp look normal – mutation didn’t affect protein – raise temp and now mutation shows a phenotypic event SEC (secretary mutants or something) – had protein stuck in the cytoplasm – can never enter the ER also had mutants stuck in the ER or the vesicles in the ER, golgi and secretary vesicles Fused yeast with another yeast – pathway is identical to that of the other dude in previous slides Powerful tool – allowed him to isolate the proteins that were mutated or he did what was called rescue – add proteins and see which will rescue the phenotype Know now what proteins are being involved Protein working in humans is like one in yeast The organelles of the secretory pathway RER ER - to Golgi transport vesicles and the intermediate compartment Golgi complex (cis, medial and trans compartments) Secretory vesicles Regulated exocytosis (regulated secretion) Need some sort of stimuli Constitutive exocytosis (continuous secretion) Secreted all the time SEE SLIDE 8 (CELL 5 SECRETION) Protein sorting Major sorting decision Synthesis on free or membrane-bound polyribosomes How does a protein enter the secretary pathway? How does it start? Gunter Mobel? Won a Nobel prize yay! mRNA in cytoplasm, ribosomes bind to it and proteins are made. Same proteins will stay in cytosol and some of them will have an ER signal sequence….. Protein sorting Protein synthesis in cytosol or on RER Signal peptide at NH2end of proteins destined for RER 5-10 hydrophobic AA NH2 Met-X-(KRH)-X-H-H-H-H-H-H-H-XXX… COOH Cotranslational import Translation begins on free ribosome; then attachment to RER Signal peptides first recognized on proteins synthesized on RER Signal sequence Right at the end terminus at the start of the protein (carboxyl group) X = any amino acid (often 19 of them) prolene can disrupt it (don’t add them) H = hydrophobic Actual sequence not important Hydrophobic need to be in a row How would you know if you have a secretary protein? Hydropathy plot Recombinant DNA technology can direct protein traffic in a cell SEE SLIDE 12 (CELL 5 SECRETION) Protein that is suppose to go to the ER – if we chop it off we can take the ER signal and put it on something that is normally in cytosol – if we put this tag on another protein that protein will go to the ER and go through secretion and the old protein will stay in the cytosol Recognitio
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