MCB 7: Intracellular Transport and Membrane Trafficking II

Question 1

What are the post-translational modifications that occur in the ER?

Answer 1

• after the polypeptide chain is fully synthesised and is translocated into the ER, it is folded into its final 3D shape
• initial glycosylation can occur: sugar chains are covalently attached to the protein
• disulfide bonds may form between certain cysteine residues
• multimeric proteins, proteins made of many sub-units, will assemble in the ER
• these proteins are then budded off into transport vesicles to the Golgi apparatus
• proteins actually take a while to fold, and sometimes they are not able to fold properly - a misfolded protein
• these misfolded proteins are exported from the ER into teh cytosol where it is degraded

Question 2

What happens to misfolded proteins in the ER?

Answer 2

• they attach themselves to chaperone proteins in the ER lumen
• then they are transported into the cytosol where they are degraded

Question 3

Give an example of a disease caused by blocked ER exit due to protein misfolding

Answer 3

Question 4

Describe the Golgi stack structure

Answer 4

• the Golgi stack has two faces: cis and trans
• the cis face is the entry face, which is next to the ER
• the trans face is the exit face, which faces the plasma membrane
• soluble proteins and lipids enter the Golgi network via transport vesicles from the cis face

Question 5

Describe how proteins are transported through the Golgi apparatus and why they can move through

Answer 5

• proteins are released from the ER through transport vesicles
• these vesicles fuse together to form the cis face of the Golgi apparatus
• as they move through the Golgi network, Golgi enzymes at specific locations modify the proteins
• the modifications are important because they add the signal sequence to direct the final location of the protein
• proteins are able to move along the apparatus because of the Cis Maturation Model:
• first, the cis cisterna becomes part of the medial cisternae
• behind it, a new cis cisterna is formed from the ER vesicles
• then one of the medial cisternae migrates and becomes a trans cisternae
• the trans Golgi network then bud off into vesicles and they migrate into their target locations (organelle, membranes)

Question 6

What kind of modifications occur in the Golgi apparatus?

Answer 6

• the Golgi apparatus is another site of carbohydrate modification
• although initial glycosylation occurs in the ER, some proteins undergo further modifications in the Golgi
• e.g. more complex oligosaccharide side chains are and/or removed
• the enzymes that need to act first are situated in the cis cisternae and enzymes that act later are in the trans cisternae
• it is a strictly ordered process

Question 7

What are some pathways involved in vesicular transport?

Answer 7

1. Outward Secretory Pathway
   - e.g. transport from Golgi apparatus to vesicles to the early endosome, then to lade endosome then to lysosome, where they actually do their job
   - we also have exocytosis, where materials are carried by transport vesicles to the plasma membrane and is released into the extracellular space
2. Inward Pathway
   - material is transported from the extracellular space into cells, usually done by endocytosis
   - vesicles pinch off from the plasma membrane and bring the material into early endosomes
   - some of the material is degraded in the lysosomes

Question 8

Define endocytosis

Answer 8

• the process by which cells take in materials through an invagination of the plasma membrane, which surrounds the material in a membrane-enclosed vesicle

Question 9

Define exocytosis

Answer 9

• the process by which materials exit the cell via fusion of a vesicle with the plasma membrane

Question 10

How do vesicles transport cargo from one compartment to another?

Answer 10

• the donor compartment is where the cargo originates from and the target (acceptor) compartment is the destination
• a vesicle containing the soluble proteins is budded off the donor compartment which then travels to the target compartment
• this fuses with the plasma membrane of the target compartment, releasing the proteins
• the membrane proteins from the donor compartment that budded off and formed onto the vesicle, is now part of the plasma membrane of the target compartment

Question 11

What are coated vesicles? Give an example

Answer 11

• vesicles that bud off from membranes have a protein coat on their cytosolic surface
• these are shed before they fuse into the target membrane
• e.g. clathrin-coated vesicles

Question 12

Describe the stages of vesicle budding (e.g. using clathrin-coated vesicles)

Answer 12

• there is a clathrin-coated pit (invagination) in the plasma membrane
• clathrin molecules assemble into a basket-like network, which then starts to shape the membrane into a vesicle that then pinches off

Question 13

Describe the role of adaptins and how vesicle budding occurs

Answer 13

1. Molecules for onwards transport have specific transport signals that are recognised by cargo receptors
   - adaptins help capture these cargo receptors by binding to them
2. a protein called dynamin assembles as a ring around the neck of the deeply invaginated coated pit, causing the ring to constrict
3. This causes the vesicle to pinch off the plasma membrane
4. After budding, the coat is shed, so that the vesicle can interact with the membrane it will fuse

Question 14

Describe how the structure of clathrin molecules allows it to form a coated vesicle

Answer 14

• a clathrin molecule is a three-legged structure called a triskelion
• the individual triskelions interact through their leg domains, forming a closed cage

Question 15

How do vesicles actually deliver their cargo to the target compartment?

Answer 15

1. Cargo Sorting and Vesicle Formation:
   - only the specific soluble proteins for transport are concentrated in area where the vesicle buds off
   - vesicles are pinched and budded off
2. Vesicle Movement
   - vesicles move along microtubules to get to the target compartment
   - they need to find the acceptor compartment membrane
3. Vesicle tethering/docking
   - vesicles have to dock on the acceptor compartment membrane of the target compartment
4. Vesicle fusion
   - vesicle fuses with the membrane and the molecules are released into the extracellular space

Question 16

Describe the fusion of vesicles with target membranes and explain how certain proteins are responsible for this

Answer 16

1. Tethering:
   - a filamentous tethering protein on membrane binds to a Rab protein on the surface of the vesicle
   - this allows the vesicle to dock on its particular target membrane
   - once the tethering protein has captured a vesicle by holding its Rab protein, SNAREs on the vesicle (called v-SNAREs) interact with complementary SNAREs on the target membrane (called t-SNAREs)
   - this firmly docks the vesicle in place
2. Fusion:
   - the same SNAREs involved in docking also play a central role in catalysing the membrane fusion required for a transport vesicle to deliver its cargo

Question 17

What are Rab proteins?

Answer 17

• they are important in making sure that transport vesicle fuses with the correct target membrane
• there are different Rab proteins, each specific for a particular type of vesicle
• each organelle and each type of vesicle carries a unique combination of Rab proteins, which serves as a molecular marker for each membrane type

Question 18

What are SNARE proteins

Answer 18

• SNARE proteins provide additional recognition during vesicular transport
• there are v-SNARE (vesicular-SNARE) on the vesicle
• there are t-SNARE (target-SNARE) on the target membrane
• these proteins are transmembrane proteins that interact with one another to facilitate fusion of the vesicle with the target membrane

Question 19

What is secretion?

Answer 19

• The process of when soluble proteins are delivered to the cell surface
• (by exocytosis)

Question 20

What are the two types of exocytosis?

Answer 20

• unregulated / constitutive secretion
• regulated secretion

Question 21

Explain the differences between unregulated/constitutive and regulated secretion of exocytosis

Answer 21

Unregulated / Consitutuive Regulation

• happens continuously, all the time
• as soon as product is made, it forms a transport vesicle and is transported and fused onto the plasma membrane for secretion
• does not have any special signals

Regulated Secretion:

• requires an extracellular signal, such as a hormone or neurotransmitter
• only happens in certain types of cells, generally cells that store secretory molecules
• the secretory vesicles store the content until they receive a signal
• then they will move to the plasma membrane and release the content

Question 22

Give an example of regulated exocytosis

Answer 22

Question 23

What are the three forms of endocytosis?

Answer 23

• pinocytosis:
• ‘cellular drinking’
• ingestion of fluid and molecules via small vesicles (<150nm in diameter)
• phagocytosis:
• ingestion of large particles (microorganisms or cell debris) via large vesicles (>250nm in diameter)
• usually by specialised cells called phagocytic cells
• receptor mediated:
• cargo binds to specialised transmembrane proteins called receptors

Question 24

Describe pinocytosis

Answer 24

• cells continuously ingest parts of their plasma membrane along with small volumes of extracellular fluid
• the rate with which different types of cells carry out pinocytosis varies
• it is mainly carried out by clathrin-coated pits and vesicles
• extracellular fluid is trapped in the coated pit as it invaginates to form a coated vesicle
• extracellular fluid is internalised and delivered to endosomes
• this fluid intake is generally balance by fluid loss by exocytosis

Question 25

Give examples of phagocytosis

Answer 25

Question 26

What does receptor mediated endocytosis allow?

Answer 26

• it allows the internalisation of material that can be concentrated many-fold (up to more than 1000 fold)
• so that minor components of extracellular fluid can be taken up without taking up correspondingly large volumes of fluid

Question 27

Describe the process of receptor mediated endocytosis using cholesterol as an example

Answer 27

In the blood, cholesterol is transported in the form of LDL (low-density lipoprotein particles)
- LDL molecules bind to LDL receptors, transmembrane proteins, on the plasma membrane

• these receptors concentrate in clathrin-coated pits
• eventually a clathrin-coated vesicle is formed
• the clathrin-coat molecules are released so the vesicle is uncoated
• the vesicle fuses with an endosome and contents are released
• the endosome has a lower pH, causing the receptor and LDL to separate
• the LDL molecules are delivered into lysosomes, where hydrolytic enzymes degrade the LDL down to form free cholesterol, as it is membrane soluble
• cholesterol in the cytosol can now be incorporated in newly synthesised plasma membranes
• the receptors are budded off into transport vesicles and are returned to the plasma membrane for reuse