1. Signaling at the Surface
Lecture 21
Vesicular Traffic
reading: Chapter 14
Signaling at the Surface
reading: Chapter 15

2. ER vesicle budding and fusion to form new cisterna
retrograde return of ER-resident proteins
cysternal progression and maturation
secretion – constitutive, regulated
vesicle transport to lysosomes to maintain structure
endocytosis – recycling, destruction, viral infection

3. GFP-tagged temperature-sensitive trafficking mutant protein
(G-protein from Vesicular Stomatitis Virus)

4. Ts mutant of VSV G protein
Progression through Golgi is tightly coupled to specific glycosylation
Ts mutant of VSV G protein

6. Isolated vesicles from two different strains provide cell-free assay for components necessary for progression from one Golgi cisterna to another

7. why is coat needed for budding?
Tension is proportional to Pressure Difference X Radius
LaPlace’s Law
T = p*r/2

10. GDP to GTP exchange

11. Sec23/Sec24
Sec13 and Sec31 proteins also participating in the coat formation are not shown
The coat does not assemble unless there is a specific cargo inside which is recognized by the signal receptor

13. What can block un-coating?
1. Mutations in Sar1 of ARF
2. Non-hydrolyzable analog of GTP (GDP-g-S)

14. The process of fusion is mediated by several GTP- or ATP-binding proteins

15. Anterograde and retrograde transport are mediated by different coat complexes

18. Cisternal Maturation

20. unidentified
coat
COPI - purple
clartin - red
AP complex- blue

24. Signaling at the cell surface (chapters 15, 16)
Binding (again…) and transmembrane signaling
Who are the ligands and who are the receptors?
G-proteins and G-protein coupled receptors
Visual cascade and Receptor adaptation

27. There are many types of receptors that regulate differentiation and development as well as every day metabolic functions.
The signal comes from the outside, the processes are triggered inside

28. Why the Kd for ligand binding does not reflect the concentration at which half-saturation of the physiological response takes place?
1. There are many more receptors on the surface than required for full response.
2. The cascade of intracellular reactions often provides a many-fold amplification.

29. Amplification cascade following activation of b-adernergic receptor
see the example of glycogen synthesis/breakdown mechanisms, figs 15-25

30. Kd = 1.4·10-8 M
Total binding has contributions from specific (high-affinity) and many non-specific (low-affinity) binding sites. When 125I - labeled insulin is bound in the presence of 100-fold excess of unlabeled insulin, only non-specific binding will be detected.

31. Binding properties are assessed in competition experiments
Kd = 5·10-5 M
natural agonist
Kd ~ 6·10-8 M
stronger agonist
Kd = 3·10-9 M
high-affinity blocker
Occupancies by the primary substrate (L1)
and by the competitor (L2)
K1,2 – binding constants (1/Kd)

32. Identification and Cloning of receptors can be done through expression of cDNA libraries in ‘clean’ cells and then probing their responses with specific ligands.
Recall the steps that are needed to prepare and screen a cDNA library 1. 2. 3. …