1. Vesicular Trafficking
Movement From the ER Through the Golgi

2. Processing in the Golgi
Oligosaccharide modifications
Sorting to exocytic vesicles, lysosomes and ER
Proteolytic processing
Sphingolipid synthesis

3. Modification of N-linked Oligosaccharides
CIS
TRANS
As glycosylated protein moves through ER, Glc is removed
In cis Golgi, some Man is removed
In medial golgi, GlcNac is added
In trans-golgi, Gal & NANA are added

4. Mannose-6-Phosphate is Lysosomal Targeting Signal

5. Proteolytic Processing
Prohormone convertases
Pre-pro-opiomelanocortin
proopiomelanocortin
Joining peptide
Corticotropin
Corticotropin-like intermediate peptide
-lipotropin
-melanotropin
-endorphin

6. On to the Golgi http://youtu.be/sIJgv8fAsSg

7. Location & morphology of the Golgi apparatus in animals
Fig
Copyright 2008 by Saunders/Elsevier. All rights reserved.

9. Transport of carrier vesicles along microtubules
Fig. 21-2
Copyright 2008 by Saunders/Elsevier. All rights reserved.

10. Protein machinery for vesicular traffic
Fig. 21-5
Copyright 2008 by Saunders/Elsevier. All rights reserved.

11. Getting to the Golgi
Anterograde and Retrograde Traffic

12. Protein trafficking pathways through the Golgi apparatus
Fig
Copyright 2008 by Saunders/Elsevier. All rights reserved.

13. Effect of lipids on bilayer thickness
Fig. 21-3
Copyright 2008 by Saunders/Elsevier. All rights reserved.

14. Lipid gradients in the membranes
of secretory pathway contributes to protein sorting
Fig. 21-4
Copyright 2008 by Saunders/Elsevier. All rights reserved.

15. Genetic Analysis of ER-Golgi Transport
Types of vesicular transport disruption
Disrupted process
brefeldin A
N-ethylmaleimide

16. General Principles Cargo selection
signals (ie amino acid sequences)
Vesicle formation (budding) from donor compartment
Vesicle/Cargo targeting
Fusion of vesicle to acceptor compartment
Most regulation is mediated by cargo- and target-compartment-specific small GTPases

17. Anterograde transport
COPII apparatus
protein complex mediating cargo selection and assembly of budding components from ER
Retrograde transport
COPI
protein complex mediating transfer of vesicles back to ER

18. Retrograde Transport Gives Golgi Compartments Dynamic Identity

19. Cargo Selection: Export Targeting Sequences
Y xnDxE – tyrosine-diacidic domain
Found in cytoplasmic tail of TM proteins

20. Cargo Selection: Just Getting the Right Stuff
Exclusion of ER resident proteins
not well understood
fidelity factors (genes)
mutations in some genes cause increased excretion of ER resident proteins
Retrieval
COPI vesicles retrieve ER proteins and return them
KDEL signal on ER lumenal proteins
KKxx-COOH on ER TM proteins (dilysine motif)

21. Vesicle formation driving by arf-family small GTPases
Sar1, Arf 1 – Arf 6, etc…
arf•GDP recruited to membrane by GEF
GDPGTP exchange allows arf N-term to interact with membrane
arf•GTP binds coat proteins
Sar1  COPII vesicles ER Golgi
Arf1  COPI vesicles GolgiER
Rab  vesicle-target membrane fusion

22. Arf GTPase cycle
Fig. 21-6
Copyright 2008 by Saunders/Elsevier. All rights reserved.

23. COP II coat assembly on the ER membrane
Fig. 21-7
Copyright 2008 by Saunders/Elsevier. All rights reserved.

24. COPII Vesicular Coat Assembly & Budding
COPII coat formation regulated by sar1 GTPase
Sar1 activated by sec12 (GEF) in ER membrane
sar1 recruits cargo receptor-coat adapter sec23/24
TM protein export signals bind sec24
sec24/23/sar1 recruit sec31/13 coat proteins
COPII vesicles form
Protein packing and conformational changes activate GAP activity of Sec23
Sar1-GDP releases sec23, disrupting sec23/24/sec13/sec31 complex
Coat formation is dynamic
Absence of sec12 in budded vesicle allows all sar1 to become sar1-GDP

25. COP I assembly on membranes
VTC & Cis-Golgi
ER resident protein
Fig. 21-9

26. Membrane coats for transport of vesicles to & from ER
Fig
Copyright 2008 by Saunders/Elsevier. All rights reserved.

27. Transport from the ER to Golgi apparatus
Fig
Copyright 2008 by Saunders/Elsevier. All rights reserved.

28. Vesicular Delivery Rab-GTPases
Recruits tether and fusion proteins into vesicles
These form a targeting complex recognized by a docking complex
targeting
v-tethers – p115
vSNAREs – synaptobrevin
docking
t-tethers – GRASP65
tSNAREs – syntaxin 1

29. Whips and Chains Tethers – SNAP receptors (aka SNARES)
Homodimeric, 15nm long coiled-coil proteins
P115
Multisubunit, rab-activating complexes
Exocyst
COG
TRAPP
SNAP receptors (aka SNARES)
Soluble NSF attachment protein (NSF = N-ethylmaleimide-sensitive factor)
v-SNARES & t-SNARES
location
R-SNARES & Q-SNARES
a.a. in interaction domains

30. Tethering and Fusion Mediated by SNAREs
SNAREs aggregated by Rab-GTPase (vSNAREs)
Vesicle buds
SNAP25-NSF interact with vesicle to dis-aggregate and prime vSNAREs for interaction with tSNAREs
AAA ATPase

31. Wiki SNARE

32. Retrieval of ER Proteins with KDEL Signal by COPI Vesicles

33. Cycle of Vesicular Transport

34. Cargo Sorting by the Trans-Golgi Network (TGN)
Specific coat proteins direct TGN vesicles to targets

35. Lysosomal Targeting of Prohydrolase by M6P and MPRs
TGN lumen
Mannose-6-Phosphate Receptors
Cation-independent (CI)
Cation dependent (CD)
cytosol

36. MPR Cytoplasmic Tail Routing Signals

37. Assembly of Clathrin on Lysosomal/Endosomal Destined TGN Vesicles

38. Lysosomal/Endosomal Targeting
MPR targeting to lysosome/endosome bound vesicles
Vesicle Coats
AP1
Adaptor proteins
-  &  adaptins
GGA
Golgi-localising, gamma-adaptin ear domain homology, ARF-binding proteins
AP3
 &  adaptins
AP2
exclusive to plasma membrane
 &  adaptins

39. Adaptor Protein Components

40. Directed Translocation of Secretory Vesicles
apical targeting in caveolin coated, lipid-raft vesicles
basolateral targeting signals
direct protein to this membrane
constitutive basolateral targeting
subsequent sorting of apical proteins
untargeted vesicles
sorting to redistribute surface proteins
tight junctions E-cadherin
tight junctions syntaxin-3

41. Proteolytic Processing of Pro-proteins in Secretory Vesicles
Cleavage of proinsulin in secretory vesicles of pancreas cells

42. Secretory Vesicles/Granules
Material in secretory vesicle aggregates due to decreased pH & divalent cations
Membrane recycling to TGN directly or via endosome reduces vesicle size

43. Regulated Secretion
Fusion of the vesicle and plasma membrane is regulated by various means
receptor-ligand interaction and second messenger signaling
depolarization of membrane
etc…