1. Extra credit for Midterm 2
Send all textbook corrections (any chapter, any type of correction) by Monday April 9th. The more corrections, the more points the class will earn.

2. Fundamentals of Cell Biology Chapter 9: The Endomembrane System and Membrane Trafficking

3. Chapter Summary: The Big Picture (1)
Chapter foci:
Define the endomembrane system and introduce the general concepts of vesicle-mediated traffic
Understand the temporal sequence of events beginning with ER and ending at PM
Explore details of Golgi function
Understand Golgi to PM pathway
The role of the endosome in sorting
The significance of the lysosome

4. Chapter Summary: The Big Picture (2)
Section topics:
The endomembrane system is a network of organelles in eukaryotic cells
Exocytosis begins in the endoplasmic reticulum
The Golgi apparatus modifies and sorts proteins in the exocytic pathway
Exocytosis ends at the plasma membrane
Endocytosis begins at the plasma membrane
The endosome sorts proteins in the endocytic pathway
Endocytosis ends at the lysosome

5. The endomembrane system is a network of organelles in eukaryotic cells
Key Concepts (1):
The endomembrane system is a set of interconnected organelles that readily exchange materials.
The primary functions of the endomembrane system are to control the export (exocytosis) and import (endocytosis) of materials to/from the extracellular space.

6. The endomembrane system is a network of organelles in eukaryotic cells
Key Concepts (2):
Membrane-bound compartments called vesicles shuttle between organelles in the endomembrane system and are responsible for carrying material from one organelle to another.
The creation, transport, targeting, and fusion of vesicles occurs in nine steps.

7. The endomembrane system controls molecular transport in/out of cell
Endocytic and exocytic pathways
Endomembrane system
Figure 09.01: The endocytic and exocytic pathways.

8. Vesicles shuttle material between organelles in the endomembrane system
Donor/acceptor compartments
Vesicle-mediated transport
Anterograde/retrograde transport = vesicle shuttling
Exocytosis, endocytosis, and fusion of vesicles
Figure 09.02: In vesicle-mediated transport, a membrane-bound vesicle buds from one compartment and fuses with another.

9. Vesicle-mediated transport - 9 steps
Figure 09.03: Vesicle-mediated transport occurs in nine steps.
Cargo selection
Budding
Scission
Uncoating
Transport
Tethering
Docking
Fusion
Disassembly

10. Vesicle-mediated transport - 9 steps
Figure 09.04: The three main types of coats involved in vesicle-mediated transport are COPI, COPII, and clathrin coats.
Cargo selection
Budding
Scission
Uncoating
Transport
Tethering
Docking
Fusion
Disassembly

11. Mneumonic Contest C B S U T T D F D D
Best mneumonic for the 9 steps wins 2 points on their choice of midterm exams (1, 2 or 3)
C B S U T T D F D D

12. Exocytosis begins in the ER
Key Concepts:
Newly synthesized endomembrane proteins are modified in the ER.
Signaling sequences in the newly-made proteins signal their secondary destinations. Some have retention signals that keep them in the ER.
Proteins that leave the ER enter the cis-Golgi network via COPII-coated vesicles.
Retrograde vesicle transport returns ER-resident proteins from the Golgi apparatus to the ER.

13. Job of exocytic pathway – sort/distribute new proteins in endomembrane system
Newly synthesized proteins begin post-translational modification as ER resident proteins help them fold properly
COPII-coated vesicles shuttle proteins from the ER to the Golgi apparatus
Resident ER proteins are retrieved from the Golgi apparatus

14. Exocytic pathway
Figure 09.05: Cargo proteins are incorporated into nascent COPII vesicles by different mechanisms.
Figure 09.06: ER-resident proteins are occasionally carried to the CGN and must be retrieved.

15. The Golgi apparatus modifies and sorts proteins in the exocytic pathway
Key Concepts (1):
The Golgi apparatus is organized into discrete compartments called cisternae. The cisternae are stacked on top of one another, and are classified as cis, medial, or trans according to their relative location within the overall Golgi structure.
Golgi-resident proteins are primarily responsible for modifying proteins undergoing exocytosis. They are retained in the Golgi apparatus by transmembrane Golgi retention sequences.

16. The Golgi apparatus modifies and sorts proteins in the exocytic pathway
Key Concepts (2):
The extreme ends of the Golgi apparatus are elaborated into long, tubular structures called the cis Golgi network and trans Golgi network.
Both Golgi networks sort proteins into vesicles targeted to different locations. The trans Golgi network is especially effective at sorting a large number of proteins into many distinct vesicle types.

17. Site for post-translational modification (PTM)
The Golgi apparatus is subdivided into cis, medial, and trans cisternae
Site for post-translational modification (PTM)
Golgi proteins retained by retention sequence
Figure 09.08: Proteins leaving the TGN can travel to at least five different destination compartments.

18. The trans Golgi network (TGN) sorts proteins exiting the Golgi apparatus
Figure 09.09: Dynamin uses GTP to regulate scission of a vesicle from a donor compartment.

19. TGN sorting mechanisms are complex
Many different “cargo selection” mechanisms
PTM
Protein aggregation
Signal receptor
Lipid raft

20. Budding of vesicles at the TGN likely occurs by several different mechanisms
3 different mechanisms have been proposed
Curvature-inducing proteins
Modification of membrane phospholipids
Phospholipid asymmetry

21. Scission of budding vesicles requires microtubule motor-based forces
Dynamin - GTPase
Figure 09.10: Examples of the golgi cisternae, rough ER (RER), and various stages of secretory granule formation. Ly = lysosome.

22. Exocytosis ends at the plasma membrane
Key Concepts:
Cells regulate the last stage of exocytosis (fusion) for most exocytic vesicles, to control when and how much material is released into the extracellular space and to control the delivery of membrane-associated proteins to the plasma membrane.
Controlled secretion is also called regulated secretion, and is under the control of signaling pathways.

23. 2 mechanisms for controlling the final steps of exocytosis
Constitutive secretion
constant
Regulated secretion
Controlled by signaling proteins
Secretory vesicles (zymogen granules)
Condensing vacuole
Figure 09.11: Transmission electron micrograph of clathrin-coated pits and vesicles at the oocyte surface.

24. Exocytosis ends at the plasma membrane
Key Concepts:
Cells regulate the last stage of exocytosis (fusion) for most exocytic vesicles, to control when and how much material is released into the extracellular space and to control the delivery of membrane-associated proteins to the plasma membrane.
Controlled secretion is also called regulated secretion, and is under the control of signaling pathways.

25. Endocytosis begins at the plasma membrane
Key Concepts:
The onset of endocytosis is most often indicated by the clustering of cargo receptors on the plasma membrane, accompanied by the assembly of a clathrin coat on the cytosolic face of the cluster. In micrographs, this structure resembles a pit in the membrane, so it is often called a coated pit.
Coated pits complete the nine steps of vesicle transport and deliver the vesicle to an organelle called the endosome.

26. Clathrin stabilizes the formation of endocytic vesicles
Figure 09.12: The role of clathrin in endocytosis.
Figure 09.13: Clathrin-coated vesicles are uncoated and tethered to early endosomes via EEA1, which binds to phosphatidylinositol-3-phosphate and Rab5.

27. The endosome sorts proteins in the endocytic pathway
Key Concepts (1):
The endosome is formed by the fusion of endocytic vesicles with specific vesicles that bud from the TGN.
The endosome sorts materials arriving from the plasma membrane; cargo receptors are recycled to the plasma membrane, while cargo remains in the endosome.

28. The endosome sorts proteins in the endocytic pathway
Key Concepts (2):
The lumen of the endosome is slightly acidic relative to the extracellular space, and this acidity is key to the sorting mechanism.
This sorting mechanism is very different from the sorting mechanisms used in the Golgi apparatus.

29. The endosome is subdivided into early and late compartments
Specific interacting molecules ensure correct vesicles fuse with vesicles from TGN or early endosomes
EEA-1,Syntaxin,Rab 5, PIP3
Proton pump proteins play central role in sorting/activation of endosomal contents
Figure 09.14: Maturation of endosomes to form lysosomes. Sorting of cell surface receptors and M6P receptors requires acidic environment provided by proton pumps.

30. Endocytosis ends at the lysosome
Key Concepts (1):
Complete degradation of endocytosed materials takes place in an organelle called the lysosome.
The lysosome is likely generated from the endosome in several ways, and requires fusing a vesicle from the TGN that contains essential proton pump proteins and digestive enzymes with the endosome.

31. Endocytosis ends at the lysosome
Key Concepts (2):
The digestive enzymes and proton pumps are carried to the endosome by receptors that recognize a mannose 6-phosphate tag on proteins in the TGN. The mannose 6-phosphate receptors are sorted and returned to the TGN, while the hydrolases and pumps remain in the endosome. The pumps are responsible for triggering the drop in pH in the endosomes.

32. Endocytosis ends at the lysosome
Key Concepts (3):
Once the plasma membrane cargo receptors and mannose 6-phosphate receptors have been removed from the endosome, it becomes progressively more acidic; when it reaches pH 5, shape changes in the hydrolases are triggered, which activate the endosomes and allow them to begin digesting cargo.
Once digested, the cargo building blocks (sugars, nucleosides, amino acids, etc.) are transported into the cytosol for reuse.

33. Maturation from endocytic vesicle to lysosome
Figure 09.15: Assembly of the mannose 6-phoshate sorting signal.

34. Figure 09.16: Digestion of cargo molecules in the lysosome.
Endogenous proteins destined for lysosome are tagged and sorted by Golgi
Figure 09.16: Digestion of cargo molecules in the lysosome.

35. pH is used in 3 ways to control endocytosis
1) the acidic environment in endosomes helps sort cargo from receptors 2) the relatively neutral pH of the ER and Golgi apparatus keeps the hydrolytic enzymes from digesting these organelles 3) the enzymes’ requirement for a strong acid environment protects the endomembrane system from digesting itself

36. Digestion in the lysosome
Proteases
Glycosidases
DNaseII
Phosphatases
Figure 09.17: Ubiquitin-mediated degradation occurs in multivesicular bodies, which are late endosomal compartments of the endocytic pathway.

37. Lysosomes can also degrade some resident organelles
“Autophagy” by autophagosome