1. Previously Bio308 Hypotheses for molecular basis of bipolar disorder Suggest problem lies in protein targeting Proteins made in cytosol (cytosolic and membrane ones) Sorting places proteins in membrane and in lumen of organelles

2. Importance of specific translocation >50% of protein made on cytosolic ribosomes are not intended to be used in the cytosol Must cross between 1 and 3 membranes to reach final destination Mis-localization can have drastic consequences—disease or death How does the cell know where to place a protein? Cellular ‘ZIP code’ Signal Sequences and Signal Patches

3. Signal sequences Need a ‘routing code’ A section of the protein’s amino acid sequence is necessary and sufficient to tell the cellular machinery where to place that protein

4. Targeting to the ER TWO methods of targeting to ER If targeted to the ER where can a protein end up? Main point of entry into the endomembrane system Minor pathway: Sec-dependent translocation Identified first in bacterial genetic screens Post translational

5. Post-translational translocation Sec- dependent

6. Co-translational translocation Major pathway: SRP-dependent translocation First identified in in vitro experiments using canine microsomes and wheat germ translation systems Co-translational CBI 12.3

7. Co-translational translocation Important components from ER: SRP- receptor, TRAM Sec61 complex (& BiP/Kar2-- sometimes) Mammals: ER translocation involves “push” Yeast: ER translocation involves “push” and “pull”

8. So the protein can now be in the ER-- *Where in the ER and then what happens?

9. ER ER proteins Where can a protein end up in the ER?How does it get there? Lumenal proteins Single transmembrane span proteins Multipass transmembrane proteins What category do our neurotransmitter and neurotransmitter receptor fall in?

10. Getting out of the ER ER Golgi enzyme involved in glycosylation Lysosomal acid hydrolase Neurotransmitter receptor Now what?

11. Vesicular traffic Secretory pathway: also method for delivering new PM proteins ER to Golgi to trans-Golgi network  then constitutive or regulated exocytosis

12. Constitutive and Regulated Exocytosis Constitutive= constant, sometimes called ‘bulk flow’ Regulated= needs additional signal to initiate fusion of vesicle with PM Constitutive does not mean ‘un-regulated’

13. Consequences of unregulated vesicular traffic Mixing of organelle contents (  won’t function correctly) Mislocalization of proteins (  won’t function correctly) Inappropriate levels of secretion (too hi or too lo) A Dead Cell

14. Vesicular traffic control How does a vesicle ‘know’ what components it should contain? How does it ‘know’ which membrane it should go to? How does it fuse when it gets there? Our neurotransmitter receptor need to go ‘through’ 5 cellular compartments before it gets to the post synaptic membrane

15. Stages of vesicle traffic 3 Stages: Budding, targeting/docking and fusion Donor Target

16. Content selection What goes inside which vesicle? Lumenal protein: Transmembrane proteins: Combination of cytosolic and lumenal proteins determine specific vesicle content

17. Budding Fig 17-58 CBI 13.1 Clathrin

18. The SNARE hypothesis Fig 17-59 V-SNARE T-SNARE Role of Rab proteins retrograde

19. Synaptic vesicle fusion VAMP Syntaxin SNAP 25 Synaptotagmin Rab3a