1. Exocytosis, Endocytosis, Endosomes and Lysosome Formation
The Endolytic Pathway

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

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

4. 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

5. 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…

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

7. MPR Cytoplasmic Tail Routing Signals

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

9. 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

10. Adaptor Protein Components

11. 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

13. Endocytic Mechanisms

14. Lysosomes and Endosomes
Early endosome
pH 6.5-6
sorting endosome
Late endosome
pH 5.5-5
Lysosome
pH 5-4.5

15. Lysosome Structure MVBs
Protein rich membranes
Lamp/Limp family of glycoproteins
Unusual lipids
lyso-bisphosphatidic acid
thought to protect lysosomal membrane lipids from action of lumenal lipases
Multivesicular bodies (late endosomes/lysosomes)
invagination of the limiting membrane
forms internal membranes
TM proteins can segregate into limiting or internal membranes
limiting membrane can be recycled, internal is not

16. Multivesicular Bodies
Vps – vacuolar protein sorting (yeast genes)
ESCRT – endosomal sorting complexes required for transport

17. Recycling Membrane Components from Endosomes
Tubulovesicular morphology
membrane concentrated tubules
bud & recycled directly & indirectly

18. Rab GTPases Regulate Endocytic Membrane Interactions
Ras related GTPase from rat brain

19. Pretty Colored Vesicles
Gold dust through the endolytic pathway
coated pits (CP)
early endosome (EE)
late endosome (LE)
lysosome (LY)

20. Some Examples of Receptor Mediated Endocytosis
Receptor recycling
LDL-LDLR
transferrin-transferrin receptor
Receptor destruction
EGF-EGFR

21. Ok, It's In the Cell. Now What
pH dependent interactions
affects protein-protein interactions in endolytic compartments
Acidification mediated by V1V0 H+ ATPase

23. Receptor-Mediated Endocytosis
Clathrin-coated pits
AP1, 2, 3, 4
heterotetramer
, γ,  subunits target
 subunits bind clathrin
 binds receptor signal sequence
clathrin triskelion
hexamer of 3 heavy & 3 light chains

24. Clathrin

25. Formation of Coated Pits
AP2-receptor interaction
Receptor clustering
Clathrin recruitment
Dynamin collar assembly
Budding
Uncoating & recycling of components

26. Coated Pit Targeting Sequences
FxNPxY
LDL receptor
YTRF
Transferrin receptor
Yxx
minimum consensus
Tyrosine residue in these contexts is necessary & sufficient to mediate AP2 interactions and subsequent clathrin assembly

27. Targeting Sequences

28. Endocytosis Kinetics Bulk rate Binding-studies & Turnover
LDLR
EGFR
CD4
channels
Endocytosis Kinetics
constitutive
Bulk rate
measurement of lipid movement
Binding-studies & Turnover
experiments designed to measure ligand-receptor interaction strength and rate of receptor/ligand destruction, synthesis and replacement
Ligand induced
Ligand dependent anchor release
resident

29. Calveolae Plasmalemmal vesicles Associated with lipid rafts
Or as they're better known…
Plasmalemmal vesicles
Associated with lipid rafts
Membrane lipid cycling
Raft-associated protein endocytosis/exocytosis
Inner leaflet proteins
acylated proteins
prenylated proteins
Outer leaflet proteins
GPI-linked
Some TM proteins that interact specifically with glycosphingolipids/cholesterol

30. Calveolae
Calveolin
high-affinity cholesterol binding protein

31. Phagocytosis

32. Macropinocytosis in Action

33. Invasive Particles Evade Degradation