Lecture 23 10 31 12 if housekeeping receptor mediated endocytosis receptors may go to Golgi before going back to plasma membrane Other vesicles may fuse with early endosome and vesicles from that compartment may exit and deliver receptors back to membrane Those vesicles may go to golgi and either go to plasma membrane or cytoplasm Other vesicles may bud from early endosome and deliver contents to intermediate compartments that eventually lead to lysosomal compartments How do vesicles end up merging with one of those membrane compartments and not any other What brings up fusion Vesicles do no move by diffusion but by cytoskeletal elements like microtubules using the motor proteins attached to them Upon reaching target compartment and will attach using tethering protein like hooks until they merge At neurosynapse vesicles with signal neurotransmitters Upon signaling will merge with membrane of neuron at terminus and then secrete signal contents into synapse triggered by change in membrane potential Process Vesicle originally brought into cell by receptor mediated endocytosis thus with a clathrin coat that has been shed Vesicle sitting in cytoplasm still with receptor and ligand attached this vesicle is meant to merge with a target membrane this is the endosome In addition to receptor and ligand the vesicle has SNARE SNARE filamentous proteins Will form complex with proteins on membrane V SNARE vesicular T SNARE target membrane When membranes are close enough the SNAREs will twist around each other to make complex and will bring vesicle and target membrane close enough to merge SNARE has G Protein active when with GTP will change conformation when GTP hydrolyzes Rab GTP Rab is G Protein When membranes are brought close together vesicle and target membrane Rab GTP will bind to filamentous tethering protein Establishes link that keeps vesicle in vicinity of target membrane Tethering protein long protein that dangles but has affinity for the target protein Once membranes are close enough T snare and V snare interact tangle twist around will form 4 subunit rope like complex where they will twist Twisting is energy releasing Spontaneous reaction to bring membranes together Water molecules will purge out squeezed out of membrane for the 2 to fuse Doesn t take energy to twist takes energy to separate Don t need to remember specific v snare and t snares Proteins involved in contortions are recycled in another cycle of membrane and target membrane fusion After merging snares form rope like complex Snare complex must be separated with energy snare dissociation NSF protein with accessory proteins like snap form complex and using ATP hydrolysis will separate v snares from t snares vesicle has just become uncoated with hsp 70 Rab GTPase Protein binds to motor protein and adaptor protein links rab to motor protein Each step hydrolyzes ATP Rab protein allows vesicle to move toward target membrane Once close enough to target membrane Rab detaches from motor protein and attaches to tethering protein T snares and v snares form complex upon becoming close enough and will fuse Rab proteins hydrolyzes GTP GDP and return to membrane and are available for another cycle V and T snares separate and become available for another cycle Various Rab proteins are specialized to handle particular membranes to handle particular routes merging with mitochondria membrane Sorting of newly synthesized proteins Globlet Cels Found in wall of intestines Will secrete proteins that have protective roles mucigens to protein from digestive enzymes and will lubricate for movement of material to minimize friction made of proteins and carbohydrate chains of sugars Will coat small intestine When secretion is triggered Vesicles of mucigen granules have lipid bilayer Will fuse to membrane to secrete to release substance Nucleus golgi complex Rough ER 1 polarized cells nucleus at one end secretory granules at another end 2 each secretory vesicle contains something different from every other organelle in the cell How does assorting take place Proteins can be made 1 ER 2 mitochondria or 3 cytoplasm George Palade to find sorting of root of assembly Blobel molecular mechanisms of sorting Both won nobel prizes Classic sorting model pancreas Pancreatic Acinar Cell Alpha cells secrete glucagon beta cells secrete insulin acinar cells release digestive enzymes are delivered to intestines One food in intestines reaches a certain cell hormone is released from cells to trigger acinar cells to release digestive enzymes released through duct duct leads to small intestine to complete digestion digestive enzymes released through duct goes to small intestine to complete digestion acinar cells line duct with their secretory vesicles sitting facing the duct secretory vesicles contain lipid bilayer and precursor enzymes Enzymes present with rough ER and golgi next to nucleus Intermediate vesicles between rough ER and secretory vesicles Cells stimulated by peptide hormone calcium level changes in cells and vesicles will fuse with plasma membrane stimulated secretion 1 More likely than not that the secretory vesicles will actually secrete primarily involved in secretory proteins 2 Take slices of pancreas placed in buffer secretions will still occur can survive in animal free environment and continue to function what route and where proteins in vesicles traced to review protein synthesis 350 360 370 3 locations of ribosomes mitochondria and chloroplasts free ribosomes RER bound ribosomes 5 to 3 end of mRNA ribosomes on 5 end will begin to translate protein and form polypeptides that tangle from large subunit Polypeptides become longer as move towards 3 end amino end is what at is end of chain coming out of large subunit thus amino end is synthesized first Made from either free ribosomes or on ER bound ribosomes Endoplasmic Reticulum Smooth ER and RER Ribosomes on RER large subunit sits directs on RER Cytoplasm bathes ribosomes Primary function of RER protein synthesis Smooth ER sometimes stores calcium Involved in detoxification Site of lipid biosynthesis What is route they follow from RER to secretory vesicle Pulse Chase Studies Palades Actual experiement Take pancreatic cell and place in Erlenmeyer with amino acids and salt to make secrete fluid Incubate with radioactive amino acids into cells Become incorporated into proteins Over time able to trace where radioactive proteins are in cell track movement from RER to secretory vesicle What is root of
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