Lecture 10/31Endosomes- move through microtubules- attach to target with tethering components- don't move to cytoplasm through diffusionTethering → docking → fusion- endocytic vesicles to target membrane- receptor mediated endocytosis into cytoplasm- loses cost goes to early endosomeProcess- g protein changes conformation when GTP → GDP (Rab Protein)- 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 recycleIn neurotransmitter ...Docking, fusion, SNARE dissociation- ATP hydrolysis separates v-SNAREs and t-SNAREsResolution- coated vesicle (green ^^^^^^^^) = clathrin- 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- v-SNAREs and t-SNAREs bind- Rab recycles- specificity comes from RabSorting of Newly Synthesized ProteinGoblet 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 intestineHow does sorting work?- George Palade (1974)◦ sorting root of assembly- Blobel (Palades student)◦ molecular mechanisms responsible for sortingSorting 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 enzymesPancreatic 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 secretionProtein Synthesis in Eukaryotes- free ribosomes (in cytoplasm)- ER-bound ribosomes- mitochondria and chloroplastsWhere are proteins that end up in secretory vesicles made?- mRNA (free ribosomes)ribosomes → AAA 3' 5' amino end of proteinbegin to form polypeptides as ribosomes move along mRNAER-bound Ribosomes (picture → ) large subunit of ribosomes sits on RER RER SER functions = source of Ca++, detoxification, lipid biosynthesisPulse – Chase Studies1) slices of pig pancreas → put in erlenmeyer flask2) incubated with radioactive amino acid → it incorporates into protein3) traced the path of newly made proteins in cellHow to distinguish between routes?- Inject dye with H2O and can map pathway through translucent tubesLecture 11/2 Lecture 11/5Lecture 11/7ER 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- enzymes that recognize sugars not really defined so we look at the best mechanism- Core Glycosylation 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 degradationDuring 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 vesiclesSorting of Lysosomal Enzymes- proteins destined to go to lysosome- enzyme N-Acetylglucosamine phosphotransferase phosphorylates mannose- enzyme phosphodiester glycosidase removes GlcNAc- identifies proteins as lysosomalMannose-6-phosphate receptor- clathrin vesicles go to endosome- receptor recycled because pH of endosome causes loss of affinity for ligand2 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- inclusionsVesicular 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 vesiclesER-bound ribosomes- secreted proteins- plasma membrane- ER- Golgi- lysosomesFree Ribosomes- cytosol- mitochondria- chloroplast- nucleoplasm-
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