Protein Sorting and Intracellular Traffic

Question 1

What is the difference between the SER and RER?

Answer 1

RER has ribosomes
SER extends throughout the cytoplasm

Question 2

What is the structure of the ER?

Answer 2

Continuous with the outer membrane of the nuclear envelope and consists of tubules extending through the cytoplasm

Question 3

Outline the function of the SER?

Answer 3

Lipid synthesis e.g. cholesterol
Synthesis of steroid hormones
Storage and release of Ca2+ (SR)
Detoxification in the liver

Question 4

Outline the function of the RER?

Answer 4

protein synthesis - processing transmembrane proteins destined for ER, golgi, plasma membrane
lysosomes, endosomes (hydrolase enzymes)
The site of protein synthesis for proteins that need to be secreted outside of the cell

Question 5

What is the difference between free ribosomes and membrane bound ribosomes?

Answer 5

Free ribosomes synthesise proteins that will be transported to the nucleus, mitochondria or used in the cytosol whereas membrane bound are for proteins in the ER lumen that will be sent to the golgi and released out of the cell

Question 6

Outline the process of co-translational translocation

Answer 6

Translation is stopped due to the presence of the ER signal sequence
The entire thing is translocated to the ER membrane where translation is resumed and the protein is threaded into the ER
Here the protein remains in its primary sequence so no chaperone proteins needed

Question 7

Outline the structure and function of Signal Recognition Particles

Answer 7

They have RNA and 6 proteins
They recognise the signal sequence
Translational pause domain binds to ribosome and stops translation
Signal sequence binding pocket binds to the signal sequence of the protein
Causes a pauses in translation
Taken to membrane where SRP binds to SRP receptor in the ER membrane protein is then threaded into the lumen via a protein translocator
mRNA is degraded and ribosomes returned to the free pool

Question 8

In what 2 ways are proteins modified in the ER?

Answer 8

1. Folded into its 3D conformation via ionic bonds, hydrogen bonds, Van der Waals attraction and disulphide bond formation
2. Glycosylation - 14 sugar oligosaccharide is added via the N terminus of an asparagine side chain

Question 9

What is the importance of glycosylation?

Answer 9

1. Quality control - (if the protein is folded properly)
2. Recognition
3. Protection

Question 10

Outline the process of quality control

Answer 10

1. If the protein is folded correctly then 3 glucose are cleaved from the chain
2. If it isn’t folded correctly then glucosyl transferase enzymes add a single glucose
3. Calnexin binds to the unfolded protein and the protein is given another chance
4. If it still isn’t folded correctly then it is degraded. Chaperon proteins are bound and ubiquitin tags are added it is taken to the proteosome and degraded

Question 11

What happens if there is an accumulation of misfolded proteins?

Answer 11

Unfolded Protein Response occurs
This inhibits protein synthesis
Degrades misfolded proteins by making more enzymes and increases transcription of chaperones
If this continues then apoptosis occurs

Question 12

What is the pulse-chase experiment and what does it show?

Answer 12

1. Addition of radiolabelled amino acids
2. All newly formed proteins are radiolabelled “pulse”
3. The cells are then returned to a non radioactive medium - “chase”

Shows that the proteins move through the cell

Question 13

What are the 3 trafficking pathways?

Answer 13

Secretory
Endocytic
Retrieval

Question 14

How does a vesicle move from one component to another?

Answer 14

Buds off of the membrane and dissociates from the donor compartments it then moves through the cytosol and fuses with the target compartment

Question 15

What are the types of vesicles?

Answer 15

COPII coated - from the ER
COPI coated - from the golgi
Clathrin coated - from the plasma membrane (endocytosis) and between golgi and endosomes

Question 16

How do vesicles ensure specificity?

Answer 16

GEF activates Rab-GDP to form Rab-GTP which is in the vesicle membrane. This will then bind to the Rab-effector protein in the target membrane causing the vesicle to tether to it

Question 17

How do membranes fuse?

Answer 17

V-SNAREs on the vesicle are specific to T-SNAREs on the target membrane. These wrap around each other to form a stable trans-SNARE complex

Question 18

What is the golgi apparatus?

Answer 18

Stacks of flattened membrane enclosed compartments termed cisternae
Has a cis face closer to the ER
Has a trans face closer to the plasma membrane

Question 19

What is the purpose of processing oligosaccharide chains in the golgi?

Answer 19

Promotes correct folding of proteins
Prevents unwanted aggregation
Acts as signals for sorting and targeting to the correct pathway

Question 20

What are the pathways of exocytosis?

Answer 20

Constitutive secretory pathway - immediate release
Regulated secretory pathway- stays within the vesicle until it is signalled to be released e.g. by a hormone

Question 21

What is the lysosome?

Answer 21

Degradative organelles with hydrolytic enzymes that digest un wanted material
Active at a pH of 4.5 which is maintained by vacuolar ATPase
Vesicles must be delivered here with clathrin coats and mannose 6 phosphate tags

Question 22

How is lysosome formation coupled to endocytosis?

Answer 22

Molecules are taken up in endocytic vesicles which fuse with early endosomes which mature to late endosomes. Transport vesicles carrying acid hydrolases from the trans- golgi network then fuse with these and mature into lysosomes

Question 23

What happens to the material in endocytic vesicles once fused with the early endosomes?

Answer 23

Can be degraded or recycled (vesicles will bud off and fuse with a recycling endosome)