1. Vesicle formation and targeting is a multi-step process
2. Formation of coated transport vesicle…
3. Targeting and docking to specific compartment…
SNAREs and Rabs
Target compartment
GTP
GDP + Pi
(ATP, GTP, and cytoplasmic protein factors…)
4. Uncoating…
1. Formation of coated buds…
GAP and Sar1
GEF and Sar1
Donor compartment
Coat proteins (“COPs” or “coatomer”)
Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport:
Formation of coated buds (requires ATP and cytosol);
Formation of coated vesicles (requires ATP and cytosol);
Transport and docking to the target membrane;
Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and
Fusion (blocked by NEM).
5. Fusion…
SNARE plus other fusion proteins

2. Lecture 16 Vesicle transport and targeting in the secretory pathway
COP coated vesicles
SNAREs
Protein sorting/targeting
Secretion - Golgi to plasma membrane
Retention in ER
Golgi to lysosome
How are proteins sorted to appropriate vesicles so that they are transported to proper location?
What are the address labels?

3. Two secretory pathways; constitutive and regulated
Default pathway for ER/Golgi proteins
If no address label, then secrete
15_28_trans_Golgi_net.jpg
ECB
Signal required to trigger secretory granule fusion
Example - neurotransmitter release
Inside lumen is equivalent to outside of cell

4. Regulated secretion
Secretory granules containing insulin in pancreatic cells
Signal for release is elevated glucose levels in blood

5. If secretion is default, how are resident ER proteins retained?
They aren’t!
Ex: BiP is a member of the HSP70 family that functions in the ER
BiP
KDEL
KKXX
KDEL-R
Constituitive secretion
Secretory granule
Regulated secretion
Plasma membrane ER
CGN
C, M, T Golgi
TGN
Outside
BiP escapes from ER and must be “retrieved” from the Golgi…
C-terminal KDEL in BiP sequence functions as retrieval signal…
KDEL-receptors in Golgi direct retrieval/recycling…
KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER…

6. Summary so far of protein targeting, revisited…
Secretion/membrane proteins
Protein targeting
Cytoplasm
RER
Signal sequence (hydrophobic a-helix)
Vesicle targeting
Golgi
Default
KDEL (soluble proteins)
KKXX (membrane proteins)
Lysosomes ?
Secretory vesicles
Plasma membrane
Default
A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.
(regulated secretion)
(constituitive secretion)
See ECB figure 14-5
Transport
Retrieval
How are proteins targeted to the lysosome?

7. How are proteins sorted to vesicles leaving TGN for lysosome?
Lecture 16
Vesicle transport and targeting in the secretory pathway
COP coated vesicles
SNAREs
Protein sorting
Secretion - Golgi to plasma membrane
Retention in ER
Golgi to lysosome
How are proteins sorted to vesicles leaving TGN for lysosome?

8. Lysosomes degrade and recycle macromolecules…
ECB 15-34
The lysosome of animal cells contains hydrolytic enzymes for degrading/recycling macomolecules. The vacuole of plant cells performs many of the functions of the animal lysosome, as well as regulating turgor pressure.
Lysosomes in plant and animal cells contain acid hydrolases (hydrolytic enzymes) for degrading/recycling macromolecules
pH of lumen is about 5 - acidic!
How are hydrolases and other proteins targeted to lysosomes?

9. I-cell disease helped decipher the signal for targeting proteins to the lysosome
Recessive mutation in single gene…
Fibroblasts of patients contain large inclusions (I-cells)…
Lysosomes lack normal complement of acid hydrolases…
All lysosomal enzymes secreted (secretion is the “default” fate for proteins in the ER-Golgi pathway)…
Lysosomal enzymes of “wild-type” (normal) cells are modified by phosphorylation of mannose on oligosaccharide (forming mannose-6-phosphate)…
Lysosomal proteins of I-cells lack M-6-P…
Lysosomal targeting signal resides in carbohydrate!

10. Mannose-6-P targets proteins from Golgi to lysosome
Cis Golgi
Network (CGN)
Trans Golgi
Network (TGN)
Addition of M6P
Transport via clathrin-coated vesicles to…
Lysosome
Clathrin coat
Uncoupling
(pH 5)
Mature
hydrolase
RER
Lysosomal hydrolase (precursor)
M6P receptor
Removal of phosphate &
proteolytic processing…
M6P receptor recycling back to Golgi
Lysosomal proteins are modified by the addition of M-6-P to their polysaccharides in the cis-Golgi. The trans-Golgi and/or TGN contains M-6-P receptors, whioch bind lysosomal proteins. These receptors are recruited into clathrin-coated pits/vesicles on the TGN, and transported to the endosome, and thence to the lysosome.
Addition of M6P to lysosomal enzymes in cis-Golgi
M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicles
Patients with I-cell disease lack phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts… secreted…

11. Protein targeting, revisited
Secretion/membrane proteins
Protein targeting
Cytoplasm
RER
Golgi
Plasma membrane
Signal sequence (hydrophobic a-helix)
Vesicle targeting
Default or signal?
KDEL (soluble proteins)
KKXX (membrane proteins)
Secretory vesicles
M6P
Default or signal?
A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.
(regulated secretion)
(constituitive secretion)
Lysosomes
See ECB figure 14-5
Transport
Retrieval
Next lecture: endocytosis and clathrin coats

12. Lecture 17 The pathways to the lysosome Phagocytosis Autophagy
Endocytosis
Endocytosis- The inward limb of membrane cycling
Pinocytosis
Clathrin coated vesicles
Receptor-mediated endocytosis

13. Three pathways to the lysosome
Phagocytosis
Endocytosis
Autophagy
ECB 15-35

14. Phagocytosis - “cell eating”
Performed by specialized “phagocytes:” WBCs
Phagocytosis - “cell eating”
Bacterium
Pseudopods
1. “Phagocytosis”
Entrapment by pseudopods
Engulfment: pseudopods fuse to internalize prey in phagosome…
Digestion: phagosome fuses with lysosome
Phagosome
Vesicles w
lysosomal enzymes
Some bacteria have evolved to evade digestion in lysosomes, and live as intracellular parasites or pathogens…
Myxobacteria tuberculosis (tuberculosis)…
Listeria monocytogenes (listeria)…
Yersinia pestis (plague)…
Lysosome
Where do vesicles with lysosomal contents come from?
What is their address label?

15. Autophagy (“Self-eating”); used to recycle worn-out organelles
1. “Phagocytosis”
Bacterium
Phagosome
Vesicles w
lysosomal enzymes
Endoplasmic
reticulum
Worn out organelle engulfed by ER
Lysosome
Worn out
mitochondrion
Autophagosome
2. “Autophagy”

16. Endocytosis: 1. “Phagocytosis” 3. “Endocytosis” 2. “Autophagy”
Pinocytosis (“cell drinking”) and “receptor-mediated” endocytosis
1. “Phagocytosis”
Bacterium
Phagosome
Early
endosome
Late
endosome
3. “Endocytosis”
Vesicles w
lysosomal enzymes
Endocytotic vesicles
Endoplasmic
reticulum
Lysosome
Worn out
mitochondrion
Autophagosome
2. “Autophagy”
Note that vesicles from TGN targeted to lysosome by M6P actually fuse with precursor vesicles/organelles to form lysosome

17. Overview of “pinocytosis” (“bulk” or “fluid-phase” endocytosis)
Fluid-phase endocytosis can be followed in live cells with fluorescent dyes
Proteinaceous coat
As many as ~2500 coated vesicles/min (~2-3% of surface area)!
Coated pit
GTP
GDP+Pi
~ 1’: early endosome
(pH~6)…
ATP
ADP+Pi H+
ATP
ADP+Pi
ATP
ADP+Pi
Coated
vesicle
Uncoating
(seconds)… H+
~ 5’: late endosome
(pH 5.5~6)…
Delivery of acid hydrolases from TGN…
ATP
ADP+Pi H+
~30’: Lysosome
(pH<5)…
Early endosome - late endosome - lysosome is a continuum

18. EM views - coated pit to coated vesicle
Coated pits coated vesicles
ECB 15-18

19. Protein coat is “geodesic” clathrin cage
Clathrin “heavy chain”
“Light chain”
3 clathrin “heavy chains”
(~ kDa)…
…plus…
3 clathrin light chains
(~40 kDa)…
…form…
“Triskelions”…
Spontaneously assemble into “geodesic” vesicle coats…
15.8-clathrin.mov

20. Components of a clathrin-coated vesicle
“cargo”
Cargo and receptors we know from COP-coated vesicles
receptors
clathrin
adaptins
membrane
ECB 15-19
Adaptins - adaptors that bind clathrin and cargo receptor, thereby regulating which cargo gets loaded into clathrin-coated vesicle

21. Pinching off of vesicles requires the protein dynamin
ECB 15-19
Coated pit
budding
“pinching off”
(dynamin)
uncoating
Assembly of coat causes pit to form due to 3D shape of clathrin coat

22. Dynamin is a GTPase GTP dynamin GDP GTPase that regulates pinching off
ECB 15-19
GTP
GDP
dynamin
GTPase that regulates pinching off
Explains why non-hydrolyzable GTP analogues block endocytosis

23. Clathrin-coated vesicles are rapidly uncoated
Dynamin
Adaptin complexes
Clathrin
“Clathrin-coated pit”
By the “clathrin-uncoating ATPase” a member of the HSP70 family of chaperones; requires ATP hydrolysis
Naked transport vesicles targeted to endosome…
Clathrin and adaptins recycled
GTP
GDP + Pi
Clathrin uncoating ATPase
ATP
ADP + Pi
Naked transport vesicle
See ECB figure 15-19
To endosome…

24. Lecture 17 Phagocytosis Autophagy Endocytosis Endocytosis Pinocytosis
Clathrin coated vesicles
Receptor-mediated endocytosis

25. How do cells take up specific macromolecules?
“Receptor-mediated endocytosis”
Example: Low-density lipoprotein (LDL), structure in which cholesterol is transported through our bodies
Lipid micelle:
~800 phospholipids…
~500 molecules of
cholesterol…
~1500 molecules of cholesterol ester
1 copy of apoprotein B…
Total mass: ~ 3 x 106 Da

26. Overview of receptor-mediated uptake of LDL
ECB 15-32
15_32_LDL_enters.jpg
Low pH of endosome (~6) causes LDL to dissociate from receptor
LDL is transferred to lysosome (fusion of vesicles from TGN)
Hydrolytic enzymes cleave LDL, releasing cholesterol to cytoplasm for continued membrane biosynthesis in smooth ER
Receptor is recycled back to surface (cycles about every 10 min!)

27. Defects in LDL endocytosis are associated with “familial hypercholesterolemia”…
Severe atherosclerosis at early age (strokes and heart attacks in pre-teens)
Excess LDL in circulating blood
LDL not properly internalized by cells
Recessive/single gene… encoding plasma membrane receptor for LDL (LDL-receptor or LDL-R)
Disease provided insight into mechanism of receptor-mediated endocytosis and identification/function of LDL-receptor
Mutations in N-terminal domain: LDL-R doesn’t bind LDL…
Mutations in C-terminal domain: LDL-R is not internalized…
What does this tell you about function of domains of LDL receptor?

28. Domains in LDL receptor
Based on MBoC (3) figure 13-53
LDL
N terminus of LDL receptor binds apoprotein B in LDL
C terminus binds adaptin
NH2
LDL-R
Plasma membrane
HOOC
Tyr
Asn
Val
Pro
Adaptin complex (four polypeptides)

29. Adaptin complex (four polypeptides)
Recruitment of LDL-R to coated pits requires an “endocytosis signal” in cytoplasmic domain
Based on MBoC (3) figure 13-53
LDL
Adaptin complex binds endocytosis signal in cytoplasmic domain of receptor:
-NPXY- (Asn-Pro-Val-Tyr) in LDL-R
LDL-R
Plasma membrane
At least three different adaptin complexes; bind different endocytosis signals on receptors
Adaptins recruit clathrin and initiate coated pit/vesicle formation
HOOC
Tyr
Asn
Endocytosis signal
Val
Pro
Adaptin complex (four polypeptides)

30. A single coated pit has many different receptors and cargos
Low density lipoprotein (LDL)
LDL-R
1,000s of receptors of many types per coated pit…
Same coated pits used for pinocytosis!

31. Summary of “receptor-mediated” endocytosis of LDL
Low density lipoprotein (LDL)
A single receptor makes hundreds of trips (~10 min/cycle)
pH ~7-.7.2
LDL-R
dynamin
GTP
GDP+Pi
Early endosome
ATP
ADP+Pi
pH ~6
pH ~7.2
ATP
ADP+Pi H+
Uncoating
(HSP70 family)
Coated
vesicle
Fusion
(Snares)
Proton pump in endosome acidifies endosome lumen causing LDL to dissociate from receptor
Cholesterol ester cleaved
Cholesterol released for use
Free cholesterol
Lysosome

32. Coats for all reasons: a summary of vesicle coats and functions
COPs:
Outbound: ER to Golgi transport, intra-Golgi, Golgi to plasma membrane
Retrograde: intra-Golgi, Golgi to ER
Endosomal: early to late/lysosome
Clathrin:
Plasma membrane to early endosome (endocytosis)
Golgi to late endosome/lysosome
Don’t worry about COPI vs II

33. Endosomes sort internalized receptors and ligands
ECB 15-33
Some ligands
Many receptors
Many ligands
Some receptors
Maternal IgG
Secreted IgA
Others
15_33_type_of_receptor.jpg
Transcytosis - movement of receptor to a different membrane from the one in which it was endocytosed

34. “Transcytosis” moves maternal IgG across epithelia
Intestinal lumen
Milk duct
IgG in milk
IgG is “secreted” across the mammary epithelium into milk by transcytosis
Receptor-mediated endocytosis from basolateral domain…
Secretion from apical membrane domain…
Apical membrane
IgG receptor
Tight junctions
Apical
Endosome
IgG is transcytosed into the neonate blood
Endocytosis from apical domain and secretion to basolateral membrane
Basolateral
Endosome
Epithelial cell
IgG receptor
IgG in blood
Polarized epithelial cells have distinct apical and basolateral endosome compartments
Basolateral membrane
Neonate blood
Maternal blood

35. Protein targeting and trafficking, finale!
Secretion/membrane proteins
Signal peptide
Nucleus
NLS: (basic)
Mitochondria
Protein targeting
ECB considers these all to be signal sequences
NES: (L-rich)
Cytoplasm
Chloroplasts
Peroxisomes
RER
Golgi
Plasma membrane
Signal sequence
Transit peptide
SKL at C term.
Vesicle targeting
KDEL (soluble proteins)
KKXX (membrane proteins)
Endosomes
M6P
Secretory vesicles
Default
A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.
(regulated secretion)
(constituitive secretion)
Endocytosis
signal
Lysosomes
Transport
Retrieval
Endocytosis: From plasma membrane to endosome to lysosome…

36. Membrane flow during exocytosis and endocytosis is a delicate balance
Golgi apparatus
Endosome ER
Lysosome
Original surface
Endocytosis internalizes membrane ~2-3% per minute…
Entire membrane is recycled in less than 1 hr…
Block endocytosis, exocytosis continues:
Block exocytosis, endocytosis continues:
membrane area grows…
membrane area shrinks…

37. Intermediate filaments:
Next lecture…
“Cytoskeleton”
Intermediate filaments:
Cell structure
Microfilaments: Muscle
Organelle transport in plants
Microtubules:
Cilia and flagella
Organelle transport in animals
ECB 1-20