Lecture 10 31 Endosomes move through microtubules attach to target with tethering components don t move to cytoplasm through diffusion Tethering docking fusion endocytic vesicles to target membrane receptor mediated endocytosis into cytoplasm loses cost goes to early endosome g protein changes conformation when GTP GDP Rab Protein Process Rab GTP binds to Rab effector filaments and protein tethers v SNARE filaments proteins brought close enough to t SNARE 1 5 nm then twisting causes vesicle to merge with target v SNARE and t SNARE are spontaneous energy releasing process water molecules purged between membranes and they fuse Rab proteins and SNAREs specific to location Rab and SNARE recycle In neurotransmitter Docking fusion SNARE dissociation ATP hydrolysis separates v SNAREs and t SNAREs coated vesicle green clathrin Resolution HSP70 causes clathrin to fall off Rab GTPase binds to motor protein and adaptor protein links the Rab protein Rab allows vesicle to move to target membrane Rab detaches from motor and binds to tethering protein Rab recycles v SNAREs and t SNAREs bind specificity comes from Rab Sorting of Newly Synthesized Protein Goblet cell found in wall of intestine secrete proteins with protective roles mucigen complex of proteins and carbs and long chains of sugar coats cell in small intestine to protect from digestive cells minimize friction polarized cells each compartment is unique what you find in each is different because of sorting secretion at top fuses with plasma membrane to dump into intestine How does sorting work George Palade 1974 sorting root of assembly Blobel Palades student molecular mechanisms responsible for sorting Sorting Newly Produced Proteins Co translational pancreatic model best model for examining sorting assembly of secretory vesicles signal sequences integral membrane proteins core glycosylation sorting lysosomal enzymes Pancreatic acinar cell alpha cells secrete glucagon beta cells secrete insulin acinar cells secrete digestive enzymes released into duct tube delivered to intestine pancreatic duct cells involved in secretory proteins can take slices of pancreas and incubate in buffer then cells can still survive and produce secretory proteins secretory side faces inside of the duct cells stimulated by peptide hormone for secretion Protein Synthesis in Eukaryotes free ribosomes in cytoplasm ER bound ribosomes mitochondria and chloroplasts Where are proteins that end up in secretory vesicles made mRNA free ribosomes AAA 3 amino end of protein begin to form polypeptides as ribosomes move along mRNA ER bound Ribosomes picture large subunit of ribosomes sits on RER ribosomes 5 RER SER functions source of Ca detoxification lipid biosynthesis Pulse Chase Studies 1 slices of pig pancreas put in erlenmeyer flask 2 3 incubated with radioactive amino acid it incorporates into protein traced the path of newly made proteins in cell How to distinguish between routes Inject dye with H2O and can map pathway through translucent tubes Lecture 11 2 Lecture 11 5 Lecture 11 7 ER Resident Proteins Lys Asp Glu Leu KDEL on carboxylic end NH2 COOH How do proteins get glycosylated Sugars associated with glycoproteins glucose mannose galactose fucose N acetylglucose amine N acetylgalactose amine N acetyl neuraminic acid Core Glycosylation enzymes that recognize sugars not really defined so we look at the best mechanism Types of Linkages N linked sugars attached to aspargine rER and Golgi O linked sugars attached to serine or threonine Golgi Core glycosylation aspargine transferred from lipid to sugar chain N linked sugars have identical groups called cores 2 sugars attached to aspargine enzyme that transfers chain of sugars aspargine gets sugars then amino acid then Ser or Thr Dolichol Phosphate rough ER membrane lipid dolichol phosphate in membrane of ER enzymes in ER and cytoplasm sugar and phosphate UDP UMP added to dolichol phosphate repeats all facing cytoplasm STEP 4 flipping occurs so sugars are facing inside of ER enzymes that do this are called flippases and use energy from ATP hydrolysis addition of glucose to dolichol phosphate then flip STEP 6 2 N acetylglucosamine 9 mannose 5 glucose 14 sugars Dolichol phosphate added to protein glucose removed end product that goes to Golgi Man 8 GlcNAc 2 if missfolding occurs enzymes remove for degradation During Synthesis of rER slide transfer of a chain of sugars to nascent protein donor dolichol pyrophosphate sugar chain trimmed while in ER further modifications in Golgi vesicles coming from ER and membrane becomes more complicated cis move through Golgi and find enzymes cis phase trans phase bud out of golgi to lysosome plasma membrane or secretory vesicles Sorting of Lysosomal Enzymes proteins destined to go to lysosome enzyme N Acetylglucosamine phosphotransferase phosphorylates mannose enzyme phosphodiester glycosidase removes GlcNAc identifies proteins as lysosomal Mannose 6 phosphate receptor clathrin vesicles go to endosome receptor recycled because pH of endosome causes loss of affinity for ligand 2 defective alleles in sorting of lysosomes causes I cell disease autosomal recessive trait lysosomal storage disorder intracytoplasmic inclusions in fibroblasts phosphorylation of mannose on lysosomal protein precursors lysosomal proteins follow secretory pathway cells can t complete degradation of substances ingested by cell die before 10 respiratory infection mental retardation inclusions Vesicular Transport Model doesn t seem to be correct Golgi cisternae are static organelles with characteristic resident enzymes molecules move cis trans through the Golgi by forward moving transport vesicles which bud from one cisterna and fuse with the next in a cis to trans direction Cisternal Maturation Model enzymes pushed back to previous phase by COPI vesicles proteins never leave each Golgi cisterna matures as it migrates outward at each stage the Golgi resident proteins that are carried forward in a cisterna are moved backward to an earlier compartment in COPI coated vesicles ER bound ribosomes secreted proteins plasma membrane ER Golgi lysosomes Free Ribosomes cytosol mitochondria chloroplast nucleoplasm peroxisomes Lecture 11 9 Mitochondria Experiment 1 reconstruction electron micrograph plastic sheets project image then stack sheets 4 compartments outer membrane inner membrane matrix intermembrane space take liver cells isolate mitochondrialput in erlenmeyer flask add radioactive amino acid 35 S meth see if the
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