Lecture 4: Membrane protein insertion

Question 1

What are some features of cell-free assay?

Answer 1

Easy to manipulate
Different mRNA
Can add or remove soluble components

Question 2

What is cell free assay used for?

Answer 2

Studying protein targeting in a test tube

Question 3

During protein insertion how is the polypeptide sequence prevented form fully entering the channel?

Answer 3

Hydrophobic stop transfer sequences stop their movement
This hold part of the polypeptide in the cytosol, the ER lumen and in the channel

Question 4

How do multi-pass transmembrane proteins enter the ER

Answer 4

Start transfer sequence acts as ER targeting signal and is held in the channel
Downstream there is a Stop transfer sequence that is also held in the channel
Both C and N terminus are outside the ER lumen

Question 5

What are the 4 ways membrane proteins can associate with the lipid bilayer

Answer 5

Transmembrane
Monolayer-associated
Lipid-linked
Protein-attached

Question 6

How are proteins folded at the ER?

Answer 6

Helped by molecular chaperones in the ER

Question 7

What is the purpose of folding proteins?

Answer 7

To make sure they fold into the right 3D conformation

Question 8

What are 2 examples of chaperones that help with folding?

Answer 8

Bip
Calnexin

Question 9

What is the chaperone BiP

Answer 9

ATPase
Binds exposed hydrophobic residues

Question 10

What is the chaperone calnexin?

Answer 10

Binds N-glycosylated proteins

Question 11

What is the 3 ways of modifying proteins in the ER?

Answer 11

1. Signal sequence cleavage
2. Disulphide bond formation (oxidation)
3. Glycosylation

Question 12

What is disulphide bond formation in the modification of proteins?

Answer 12

Disulphide bonds formed by oxidation of cysteine side chains
They stabilise the folded structure
Catalysed by protein disulphide isomerise and only form in the ER lumen

Question 13

What types of proteins specifically need stabilisation?

Answer 13

Secreted proteins
Exist under harsher environments

Question 14

N-linked glycosylation

Answer 14

N : Asparagine
An oligosaccharide is transferred onto the protein from a special lipid donor : dolichol
The transfer is catalysed by oligosaccharyl transferase
This occurs while the protein is coming out of the Sec61 translocator

Question 15

What are the functions of N-glycosylation in protein modification?

Answer 15

Assists protein folding
Can be modified to act as a lysosome sorting sequence
Act as ligand for cell-cell recognition events

Question 16

What is the role of N-glycosylation in the glyycocalyx?

Answer 16

Eukaryotic cells are coated in carbohydrates attached to proteins and lipids called the glycocalyx
Forms protective layer outside cell
Made at ER and golgi

Question 17

What is the role of quality control in the ER?

Answer 17

Prevent misfolded proteins from leaving the ER

Question 18

What is the process of quality control?

Answer 18

1. Misfolded proteins activate the sensor protein in the wall of the ER lumen
2. Activates chaperone genes
3. Bind to proteins and properly fold them