Conference on RER, Golgi, and secretion Objectives of the conference on Secretion
To reinforce the pathway of production and secretion of proteins of cells by considering defects in the pathway To use techniques we have discussed and introduce a new technique to isolate specific proteins to solve problems and predict outcomes of defects in the pathway To reinforce the idea that each step of a pathway is an opportunity for a disease DO THE : PULSE CHASE EXPERIMENT LOOK FOR THE : TEMPORAL APPEARANCE OF SILVER GRAINS IN THE PHOTOGRAPHIC EMULSION OVERORGANELLES OF INTEREST CELL FRACTIONATION GRIND CELL INTO COMPONENTS, SEPARATE COMPONENTSON SUCROSE GRADIENT BY ULTRA CENTRIFUGATION, AND ANALIZE COMPONENTS FOR RADIOACTIVTY TEMPORAL APPEARANCE OF RADIOACTIVE PROTEINS IN DIFFERENT ORGANELLES
SEEN BY: CELL FRACTIONATION AUTORADIOGRAPHY Immunoprecipitation Method to render specific soluble proteins insoluble when centrifuged Allows separation/isolation/identification of specific proteins in solution Antibodies are specific for the protein of interest and when bound to beads add weight to the protein that allows it to precipitate under centrifugation Website for this image File: Co-Immunoprecipitation.svg commons.wikimedia.org EVIDENCE FOR PROTEIN PATHWAY
TEMPORAL APPEARANCE OF RADIOACTIVE PROTEINS IN DIFFERENT ORGANELLES (produced from radioactive precursors e.g., labeled AA) Detected by: AUTORADIOGRAPHY - VISUAL CELL FRACTIONATION BIOCHEMICAL REACTIONS SCALER REACTIONS A + B = C VECTORAL REACTIONS A + B = C
membranes PROTEIN SORTING summary
If SIGNAL PEPTIDEto RER and start transfer SIGNAL RECOGNITION PARTICLE RIBOPHORIN - ON RER, BINDS RIBOSOME TO RER STOP TRANSFER HYDROPHOBIC AMINO ACID SEQUENCE INSERTED INTO MEMBRANE Cytosolic proteins (especially in high concentrations) can take the constitutive-like mode in that cytosolic proteins can participate in Apical secretion. LYSOSOMAL PATHWAY MANNOSE-6-PO4 DIRECTS VESICLES WITH POLYPEPTIDE TO LYSOSOMES GOLGI - POLARIZED SHAPE AND FUNCTION
CIS CONVEX REGION - PHOSPHATE GROUPS ADDED MIDDLE MANNOSE REMOVED N-acetylglucosamine TRANS CONCAVE REGION SIALIC ACID, FUCOSE ADDED GOLGI ADDS FATTY ACIDS, SULFATE GROUPS, GALACTOSE
ALSO ADDS FATTY ACIDS, SULFATE GROUPS, GALACTOSE RECYCLING OF MEMBRANE Considerations How are proteins segregated to stay in the cytosol vs enter the cisternae of RER? How is protein separated from its signal to become free in the cisternae of RER? How might the function of a signal peptidase (that must bind to a specific site on the protein where it cuts) be rendered inactive? Considerations cont How might immunoprecipitation facilitate identification of specific proteins trapped in organelles in conjunction with biochemical procedures like cell fractionation? What is the default secretion method of proteins? Considerations cont Which parts of the secretory path are required to add CHO to the proteins? Objectives of the conference on Secretion
To reinforce the pathway of production and secretion of proteins of cells by considering defects in the pathway To use techniques we have discussed and introduce a new technique to isolate specific proteins to solve problems and predict outcomes of defects in the pathway To reinforce the idea that each step of a pathway is an opportunity for a disease