protein modification and quality control

Question 1

what are the functions of the endoplasmic reticulum?

Answer 1

• insertion of proteins into membranes
• formation of S-S bonds
• proper folding of proteins
• glycosylation
• specific proteolytic cleavage
• assembly of multi-subunit proteins

Question 2

where and how do disulphide bonds form?

Answer 2

occurs specifically in the lumen of the endoplasmic reticulum. disulphide bonds are formed when two cysteine residues become oxidised to form a covalent bond

Question 3

which enzyme catalyses disulphide bond formation in the endoplasmic reticulum?

Answer 3

protein disulphide isomerase

Question 4

what are the functions of PDI?

Answer 4

can allow disulphide bonds to form in the target protein

can rearrange disulphide bonds into the correct conformation

Question 5

what is the purpose of pepetidyl-prolyl isomerases?

Answer 5

accelerate the interconversion of cis and trans isomers of proline residues

Question 6

what is N-linked glycosylation?

Answer 6

addition of oligosaccharides to the amino group of asparagine residues. occurs in the endoplasmic reticulum

Question 7

what is the oligosaccharide that is added to the polypeptide in N-linked glycosylation?

Answer 7

two N-acetylglucosamines
nine mannose sugars
three glucose sugars

Question 8

outline the process of assembly of the oligosaccharide for N-linked glycosylation

Answer 8

1- phosphate of UDP is added to the dolichol with N-acetylglucosamine
2- sugar residues are added individyally
3- five mannose sugars are delivered as GDP-mannose sugars
4- when there are five mannose sugars exposed to the cytoplasmic face, the dolichol is flipped into the membrane
5- subsequent mannose sugars are delivered one at a time, carried by dolichol phosphate

Question 9

how are precursor oligosaccharides transferred to proteins in N-linked glycosylation?

Answer 9

oligosaccharide transferase

Question 10

what is the importance of glucose trimming after N-linked glycosylation?

Answer 10

quality control mechanism
-unless the oligo is correctly trimmed, the protein will be trapped in the endoplasmic reticulum and will not move to the Golgi

Question 11

what is the purpose of chaperone proteins?

Answer 11

• assist protein translocation
• retain unfolded proteins in the ER
• mediate the unfolded protein response

Question 12

what is BIP?

Answer 12

binding immunoglobulin protein

binds to exposed amino acid sequences that would normally be buried on the exterior of a folded protein

Question 13

what is calnexin?

Answer 13

an ER membrane associated protein that binds to oligosaccharides on incompletely folded proteins

Question 14

what is calreticulin?

Answer 14

a soluble ER protein that binds to oligosaccharides on incorrectly folded proteins

Question 15

outline how calnexin/calreticulin mediate quality control in the ER

Answer 15

1- unfolded protein has glucose sugars removed by a glucosidase
2- when the protein has one glucose sugar left, it triggers binding of calnexin or calreticulin
3- once bound to calnexin, a glucosidase moves the last glucose
4- if the protein is correctly folded, it exits the ER and passes to cis-golgi
5- if not correctly folded, glucosyl-transferase adds a sugar, causing it to bind calnexin again

Question 16

what happens if there are problems with protein folding?

Answer 16

• proteins might be trapped in the wrong conformation

- proteins may be incorrectly associated with other subunits

Question 17

what is the unfolded protein response?

Answer 17

a process which allows the cell to produce more chaperones and unfolding catalysts in response to high levels of unfolded protein

Question 18

explain how BIP and Ire1 respond to unfolded protein

Answer 18

BIP is normally bound to Ire1 to keep it in the inactive
form
when there is high levels of unfolded protein, BIP dissociates
this allows Ire1 to dimerise, activating it and allowing it to exert endonuclease activity on mRNA, this produces a mature mRNA and acts as a transcription factor to produce more chaperones

Question 19

what does PERK do?

Answer 19

it is a kinase resident in the ER membrane that dimerises in stressed cells to phosphorylate eIF2, inactivating it and reducing protein synthesis

Question 20

what happens to misfolded proteins that cannot be refolded?

Answer 20

they are bound by chaperone proteins and translocated out of the endoplasmic reticulum

Question 21

what is ubiquitin?

Answer 21

a 76 amino acid protein that attaches to lysine. polyubiquitination targets proteins for proteasome-mediated degradation

Question 22

what is the Golgi?

Answer 22

membrane bound sacs known as cisternae. each cisterna carries Golgi enzymes which help modify cargo proteins

Question 23

what are the five functional regions of the Golgi apparatus?

Answer 23

cis Golgi network, cis-golgi, medial Golgi, trans-golgi, trans-golgi network