1. Getting things where they need to go: Protein Targeting Translation: Converting nucleotide sequence to amino acid chain Role of tRNA, base pairing and wobble Role of ribosome (A, P, E sites) CBI 6.4 CBI 6.5

2. Cell Neurotransmitters: synthesis and packaging Where are neurotransmitters/neuropeptides synthesized? What must happen before they can be used? packaging transport Barriers to packaging Biological membrane of vesicle Polar nature of neurotransmitter How are the barriers overcome?

3. Carrier Proteins Why are they needed? How do they work? What kind of energy is needed? Main Classes: Passive versus Active Transport Going with or against the flow Types of active transport: Coupled– ex. symports or antiports Pumps– like STE6, mdr, Ca ++ pump (ATP hydrolysis) Light driven pumps (primarily bacterial) Which class(es) likely to be used in initial packaging of neurotransmitters? In their re-uptake?

4. Neurotransmitter receptor: synthesis and packaging Is a neurotransmitter receptor a cytosolic protein? Cell Where is it synthesized? How does it get into a membrane? ? ?

5. Cytosolic vs. Noncytosolic proteins The catecholamine theory of affective disorder What sorts of situations could result in this condition? (what would alter the amount of signaling at a synapse?) 1) Don’t make enough neurotransmitter 2) Make it but don’t package it into vesicles or don’t release it correctly 3) Make/ release but receptor not present on post synaptic cell or not functioning correctly 4) Make/ Release/ Receptor there but overactive re-uptake reduces the ‘effective’ amount of neurotransmitter

6. 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

7. Signal sequences How are these signals used?Necessary and sufficient

8. 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

9. Post-translational translocation Sec- dependent

10. 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

11. 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”

12. 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?