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Conference on RER, Golgi, and secretion

Explore defects in protein secretion pathways, new isolation techniques, and disease prediction. Learn about autoradiography and immunoprecipitation methods for protein analysis.

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Conference on RER, Golgi, and secretion

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  1. Conference on RER, Golgi, and secretion

  2. Objectives of theconference 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

  3. DO THE : PULSE – CHASE EXPERIMENT LOOK FOR THE : TEMPORAL APPEARANCE OF SILVER GRAINS IN THE PHOTOGRAPHIC EMULSION OVER ORGANELLES OF INTEREST

  4. CELL FRACTIONATION GRIND CELL INTO COMPONENTS, SEPARATE COMPONENTS ON SUCROSE GRADIENT BY ULTRA CENTRIFUGATION, AND ANALIZE COMPONENTS FOR RADIOACTIVTY

  5. TEMPORAL APPEARANCE OF RADIOACTIVE PROTEINS IN DIFFERENT ORGANELLES SEEN BY: CELL FRACTIONATION AUTORADIOGRAPHY

  6. 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

  7. 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

  8. REACTIONS • SCALER REACTIONS A + B = C • VECTORAL REACTIONS A + B = C membranes

  9. PROTEIN SORTING summary • If SIGNAL PEPTIDE to RER and start transfer • SIGNAL RECOGNITION PARTICLE • RIBOPHORIN - ON RER, BINDS RIBOSOME TO RER • STOP TRANSFER • HYDROPHOBIC AMINO ACID SEQUENCE – INSERTED INTO MEMBRANE

  10. Cytosolic proteins (especially in high concentrations) can take the constitutive-like mode in that cytosolic proteins can participate in Apical secretion.

  11. LYSOSOMAL PATHWAY • MANNOSE-6-PO4 DIRECTS VESICLES WITH POLYPEPTIDE TO LYSOSOMES

  12. GOLGI - POLARIZED SHAPE AND FUNCTION • CIS • CONVEX REGION - PHOSPHATE GROUPS ADDED • MIDDLE • MANNOSE REMOVED • N-acetylglucosamine • TRANS • CONCAVE REGION – SIALIC ACID, FUCOSE ADDED

  13. GOLGI ALSO ADDS FATTY ACIDS, SULFATE GROUPS, GALACTOSE RECYCLING OF MEMBRANE

  14. 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?

  15. Considerations con’t • 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?

  16. Considerations con’t • Which parts of the secretory path are required to add CHO to the proteins?

  17. Objectives of theconference 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

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