The Endomembrane System Membrane organelles – a dynamic, integrated network

1. Protein Synthesis and Transport within the Cell • The Endomembrane System • Membrane organelles – a dynamic, integrated network • Materials are shuttled – small transport vesicles • Bud from a donor membrane compartment • Move in a directed manner • Pulled by motor proteins • Tracks – microtubules of the cytoskeleton • At their destination – fuse with membrane of acceptor compartment • Repeated cycles of budding and fusion

2. Protein Synthesis and Transport within the Cell • The Endomembrane System

3. Protein Synthesis and Transport within the Cell • The Endomembrane System • Typical biosynthetic pathway • Proteins synthesised in the endoplasmic reticulum • Modified through the Golgi complex • Transported to destination • Also a secretory pathway

4. Protein Synthesis and Transport within the Cell • The Endomembrane System • Secretory activities – two types • Constituitive • Transported from site of synthesis and secreted continuously • Regulated • Materials to be secreted are stored in large, densely packed, membrane-bound secretory granules • Discharged following specific stimulus • Examples: endocrine cells, acinar cells, nerve cells

5. Protein Synthesis and Transport within the Cell • The Endomembrane System • Materials moved out by secretory pathways • Materials moved in by endocyte pathway • From outside the cells to endosomes / lysosomes • Secreted Proteins – targeted by sorting signals • In the aa sequence or • Attached oligosaccharide

6. Protein Synthesis and Transport within the Cell • The Endomembrane System • Autoradiography • Example: Acinar cells – synthesis of digestive enzymes

7. Protein Synthesis and Transport within the Cell • The Endomembrane System • Autoradiography • Cells overlayed with photographic film • Exposed to radioisotopes in the sample • Radiolabeled aa

8. Protein Synthesis and Transport within the Cell • The Endomembrane System • Autoradiography • Tissues – washed and transferred to unlabelled aa • “pulse-chase”

9. Protein Synthesis and Transport within the Cell • The Endomembrane System • Biochemical analysis of subcellular fractions • Technique to homogenize cells • Cytoplasmic membranes fragmented and form vesicles • Types separated using cell fractionation • ER and Golgi – form microsomes • Fractioned into rough and smooth

10. Protein Synthesis and Transport within the Cell • The Endomembrane System • The Endoplasmic Reticulum (ER) • Two categories • Rough (RER) • Smooth (SER) • Composition of luminal or cisternal space unique • RER – ribosomes bound to cytosolic surface • SER – no ribosomes • RER & SER – many complex differences

11. Protein Synthesis and Transport within the Cell • The Endomembrane System • RER vs SER • RER • Extensive • Interconnected sacs – cisternae • Continuous with nuclear envelope • SER • Tubular • Interconnecting pipelines

12. Protein Synthesis and Transport within the Cell • The Endomembrane System • SER functions • Skeletal muscle • Kidney tubules • Steriod producing cells • Liver • Detoxification • Glucose-6-phosphatase

13. Protein Synthesis and Transport within the Cell • The Endomembrane System • RER functions – Protein Synthesis • Polypeptides synthesized at two locales • 1. On ribosomes attached to cytosolic surface of RER • Proteins secreted from the cell • Integral membrane proteins • Soluble proteins within ER, Golgi, lysosomes, endosomes

14. Protein Synthesis and Transport within the Cell • The Endomembrane System • RER functions – Protein Synthesis • Polypeptides synthesized at two locales • 2. Other polypeptides – on free ribosomes • Released into the cytosol • Enzymes • Peripheral proteins – plasma membranes • Proteins nucleus • Protein mitochondria, peroxisomes

15. Protein Synthesis and Transport within the Cell • RER functions – Protein Synthesis • How are proteins synthesized at different sites? • Secretory proteins – signal sequence • Polypeptide moves to cisternal space of ER • cotranslationally

16. Protein Synthesis and Transport within the Cell • RER functions – Protein Synthesis • Begins after a messenger RNA binds to a free ribosome

17. Protein Synthesis and Transport within the Cell • RER functions – Protein Synthesis • Other points to note • Interaction between signal sequence and SRP – a key step • SRP is the tag – enables specific binding of the complex to the ER • Binding through at least two interactions • SRP – SRP receptor • Ribosome and translocon • Following binding • Nascent peptide released from SRP • Inserted into translocon • On termination • Ribsome released • Translocon channel reverts to narrow conformation

18. Protein Synthesis and Transport within the Cell • RER functions – Protein Synthesis

19. Protein Synthesis and Transport within the Cell • RER functions – Processing of proteins • Polypeptide is moved from the translocon to the cisterna • Enzymes in the membrane • Signal peptide removed – signal peptidase • Carbohydrates added – oligosaccharyltransferase

20. Protein Synthesis and Transport within the Cell • RER functions – Processing of proteins • In the lumen • Molecular chaperones – BiP (binding protein) • Recognize and bind unfolded proteins • Aid in attaining native conformation • Movement of the protein into the ER lumen • Protein disulfide isomerase (PDI)

21. Protein Synthesis and Transport within the Cell • RER functions – Processing of proteins • Misfolded proteins • Transported to cytosol • Destroyed in proteasomes • Quality control