to/from the Golgi apparatus

Question 1

What is
1. ERES
2. COPII/VTCs
3. ERGIC

Answer 1

1. The cargo carrying vesicles that buds off from specific sites on the RER. ERES = ER exit site
2. Coat protein complex: Coated vesicles that are involved in cargo selection and budding. COPII undergoes fusion to form vesicular tubular clusters (VTCs), microtubules are involved in vesicle transport.
3. A more stable type of ER-and-golgi intermediate compartment.
   All of these are transport transition points that provide quality check stations.

Question 2

What is vesicle trafficking?

Answer 2

The term given to transport vesicles carrying soluble proteins and membrane between compartments.

Question 3

What are vesicles made of?

Answer 3

COP II proteins form the coat of the vesicles, Sare1 proteins act as a molecular switch and sec proteins.

Question 4

What are the characteristics and function of sare1 proteins in vesicle trafficking?

Answer 4

A monomeric GTPase protein which means it will catalyse the hydrolysis of GTP.
Sare1’s activity is regulated by an exchange factor, SEC 12. When it is GDP bound it is off and when a new GTP binds with the aid of a GTPase activating protein it turns on. Sar 1 regulates the assembly or disassembly of COPII coat proteins.

Question 5

How is sar1 recruited to the RER membrane?

Answer 5

Sec 12 interacts with GDP bound sar1 catalysing the nucleotide exchange, exchanging the GDP to GTP recruiting the GTP bound sar1 to the membrane.

Question 6

How is cargo loaded into in forming vesicles?

Answer 6

At the membrane sar1 will recruit the dimer sec23-sec24 proteins that form the inner coat of a vesicle. The sec 24 subunit binds the cargo proteins. This complex is termed the pre-budding complex. This event is not singular a vesicle is comprised of many of these complexes associating together

Question 7

How does the outer vesicle membrane form?

Answer 7

The accumulation of pre-budding complexes causes the membrane to deform at this point 2 sec13-sec31’s start associating forming hetero-tetramers that together is the COPII coat that bind to the outside of the pre-budding complex collecting it.

Question 8

When does fission of the vesicle from the membrane occur?

Answer 8

Once COPII is fully assembled the vesicle fissures from the RER membrane with the bulk soluble cargo sealed inside.

Question 9

What is the destination for COPII coated vesicles?

Answer 9

The golgi cisternae.

Question 10

What is the function of RAB proteins, tethering proteins and the two types of SNARE proteins?

Answer 10

They are all involved at different stages in the tethering, docking and then fusion of a vesicle to the target membrane from the cytosol.
Rab is a vesicle membrane bound, lipid anchor protein, that interacts with tethering protein that protrudes from the target membrane. This interaction starts the ‘docking’ and the tether protein pulls the vesicle close to target membrane. At this point v-SNARE protein (attached to the vesicle) starts interacting with the t-SNARE protein (attached to the target membrane). The SNARE proteins rachet the vesicle by intertwining with each other into the target membrane allowing them to fuse via their lipid bilayers.

Question 11

How does retrograde trafficking occur?

Answer 11

Retrograde trafficking: vesicle trafficking of not wanted cargo back to the ER from the golgi.
Instead of COPII there is COPI. The process is essentially the same as anterograde trafficking but with slightly different proteins. The proteins are still activated by GDP GTP exchange. The main difference in the process is that the COPI complex is made and put in place all together as oppose to the subunits being recruited to the pre-budding complex.

Question 12

What are the different compartments of the golgi apparatus end to end?

Answer 12

ER->
-cis golgi network
-cis cisterna
-medial cisterna
- trans cisterna
-trans golgi network

Question 13

How are proteins modified in the golgi?

Answer 13

• N-linked glycosylation: via asparagine
• O-linked glycosylation: via the hydroxyl groups serine threonine

Question 14

What is constitutive and regulated secretion?

Answer 14

Constitutive secretion: vesicles move directly from the golgi to the plasma membrane. This is an unregulated naturally occurring, genralised mechanism
Regulated secretion: the fusion of the vesicle to the plasma membrane is dependent on a signal. This tightly managed by a complex series of events triggered by specific stimuli such as a hormone or neurotransmitter.
These are both mechanisms for exporting cargo from the cell.

Question 15

What is exocytosis?

Answer 15

The process of exporting cargo to the exterior of the cell from the cytosol/vacuole.
This can be done by constitutive (macrophages) or regulated (insulin release) secretion.

Question 16

How does exocytosis of insulin occur?

Answer 16

Insulin is secreted from the beta cells of the pancreas.
1. Fed state = high glucose concentration in the blood -> beta cell membrane glut 2 receptors are triggered and the channel opens allowing glucose into the cell.
2. Glucose is phosphorylated glucose kinase making glucose 6-phosphate which then enters glycolysis pathway this in turn increase the cellular concentration of ATP and decreases ADP concentration.
3. ATP inhibits K+ channels stopping the ions leaving the cell. This leads to localised depolarisation across the cell membrane which in turn opens voltage gated Ca2+ ion channels creating an influx of Ca ions.
4. The SNARE proteins on insulin containing vesicles within the cell are activated pulling the vesicle to the b cell membrane. The vesicles are fused with the membrane by the vesicle SNARE intertwining with the cell membrane’s SNARE and insulin is ejected from the cell.
This is an example of regulated secretion.

Question 17

What is a lysosome?

Answer 17

Plasma membrane bound sacs of hydrolytic enzymes that digest extracellular material (endocytosis and phagocytosis) and worn out cellular material (autophagy).

Question 18

What is endocytosis, phagocytosis and autophagy?

Answer 18

Endocytosis = the general term given for when cells engulf substances from their environment via vesicles and ingests them. import into cell -> early to late endosome -> lysosome
Phagocytosis = a specialised form of endocytosis for large particles such as bacteria, dead cells or cellular debris and is used by specific cells namely phagocytes (immune cells) such as macrophages. import into cell -> phagosome -> lysosome
Autophagy = the degradation of cytoplasmic organelles, proteins and macromolecules. particle already in cell -> autophagosome -> lysosome

Question 19

How are hydrolytic enzymes transported safely to forming lysosomes?

Answer 19

Hydrolytic enzymes are dangerous to cellular contents but they are only active at acidic pH. The cytosol has a ~ neutral pH. Lysosomes create a ~pH 5 via an H+ pump that is driven by an ATP hydrolysis pump. This pumps protons into the lysosome making it acidic. The dangerous enzymes are transported as safe precursor by having an extra peptide bound to them. These types of enzymes are called zymogens. The peptide is removed from the zymogens by the acidic conditions in the lysosome.

Question 20

How are materials transported to the lysosome?

Answer 20

Protein arrives from the ER to the cis golgi network. The protein travels through the different parts of the golgi and is modified multiple times at the different compartments by N or O linked glycosylation. As the protein travels through the golgi network part of an attached oligosaccharide is ‘pruned’ which reveals mannose-6-phosphate. M6P acts as a sorting signal and binds to an M6P receptor in the trans golgi network. A clathrin coats the membrane in proximity to the M6P receptor starting the budding. When vesicle membrane fuses and leaves the golgi clathrin dissociates. The linking of clathrin to cargo is facilitated by adaptin 1 protein.

Question 21

How are budding vesicles fissured from the golgi membrane?

Answer 21

The clathrin coat creates a lattice of pentagons around the budding vesicle. Dynamin polymerises around the ‘neck’ of vesicle still attached to golgi. It forms in a spiral that undergoes a conformational change driven by GTP hydrolysis that extends the spiral separating the vesicle from the golgi.

Question 22

Other than the golgi when are clathrin coats formed around vesicles?

Answer 22

During clathrin mediated non-phagocytotic endocytosis, when the vesicles are budding at the plasma membrane to enter the cell.

Question 23

Which protein mediates the transport of microorganisms across the plasma membrane into a cell.

Answer 23

Cytoskeletal actin molecules for phagocytosis of the microorganism.

Question 24

How do buds form during clathrin mediated endocytosis?

Answer 24

Cargo receptors spanning plasma membrane will recruit and bind the selected extracellular cargo. After which, adaptin 2 proteins will bind to the cytoplasmic tails of the cargo receptors. The bound adaptins will recruit a clathrin each. The clathrins then start associating with each other starting the budding. This followed by dynamin polymerisation and the vesicle forming with the dissociation of the clathrin coat

Question 25

What happens after a vesicle is formed during endocytosis?

Answer 25

It fuses with an endosome where the materials in the bulk cargo are separated and sorted. The constituents are then either
- transported to a lysosome for degradation
-transported to back to the plasma membrane and recycled
- or undergo transcytosis where its transported to an adjacent cell

Question 26

How does the endosome ‘decision making’ process occur?

Answer 26

It depends on the receptor. If the ligand is bound for a recycling receptor -> plasma membrane if it is a transcytosis receptor it will go to the adjacent cell.
If ligand and receptor dissociate -> lysosome

Question 27

What is an LDL receptor and what does it do?

Answer 27

A trans plasma membrane protein that binds to LDLs (low density lipoproteins that help package cholesterol) outside the cell and adaptins inside the cell -> adaptins bind clathrin internalising LDL (bad cholesterol) into coated vesicles -> transport to lysosomes

Question 28

What do transferrin receptors do?

Answer 28

They interact with ferrotransferrin which is essentially a trap for Fe ions -> together they form a vesicle mediated by clathrin and adaptin -> transport to endosome -> Fe ions released into cytosol due endosome acidity and the now apotransfferrin and receptor are recycled at cell surface.

Question 29

What do EGF receptors do?

Answer 29

EGF = epidermal growth factor
Epidermal cell growth can be switched on and off by EGF
To switch on:
EGF binds to receptor -> receptor dimerises and undergoes autophosphorylation -> starts a big signal cascade leading to cell proliferation.