Hettema lecture 4

Question 1

Give 3 reasons why proteins undergo glycosylation?

Answer 1

• promote protein folding
• stabilise secretory proteins
• involved in cel-cell interactions

Question 2

What drug inhibits protein glycosylation of haemagglutinin?

Answer 2

tuicamycin

Question 3

Which residue is involved in N-linked glycosylation?

Answer 3

asparagine

Question 4

At which point is the precursor molecule attached to the unfolded protein during N-linked oligossacharide processing?

Answer 4

precursor attached to unfolded protein cotranslationally

Question 5

Which molecule is involved in disulphide bond formation in the ER?

Answer 5

PDI - can be oxidised/reduced

Question 6

Give the names of the proteins involved in aiding protein folding in the ER?

Answer 6

Chaperones -> Hsp70 / BIP (in ER)

Question 7

How are different targeting sequences added to each end of the a.a. sequence?

Answer 7

Alternative splicing generates targeting sequences either at N/C terminus

Question 8

Describe the process of protein import into the mitochondrial matrix (6 steps ish)

Answer 8

1 - unfolded precursor in cytosol is kept unfolded by Hsp70 proteins
2 - targeting sequence binds to mitochondrial outer membrane receptor
3 - polypeptide inserted into outer membrane translocon
4 - simultaneously threads through inner membrane translocon (lined up with each other)
5 - Hsp70 within matrix pulls the polypeptide through (energy provided by ATP hydrolysis)
6 - protein folds into active conformation

Question 9

Describe the process that was used to show that only UNFOLDED proteins could enter the mitochondrial matrix

Answer 9

an unfolded DHFR enzyme sequence can enter the matrix easily - with the correct MTS
however, adding MTX (methotrexate - an inhibitor) binds DHFR and locks it in a folded conformation. This prevents the crossing of the folded protein across both the inner and outer membrane

Question 10

Does mitochondrial import occur post-translationally/co-translationally?

Answer 10

Post-translational

Question 11

What is the structure of the MTS and where is it located?

Answer 11

N-terminus
20-50 a.a.
amphipathic helix

Question 12

What is the structure of the PTS and where is it located?

Answer 12

C-terminus

3 a.a. (SER-LEU-LYS-COOH)

Question 13

At what point of the process involving the import of folded proteins into the peroxisomal matrix is ATP required?

Answer 13

ATP hydrolysis occurs after the protein has been imported into the matrix. ATP released causes the receptor to dissociate from the plasma membrane and to be recycled to go and pick up more folded proteins

Question 14

What is the structure of the ERTS and where is it located?

Answer 14

N-terminus
core of 6-12 hydrophobic a.a
preceded by a few positively charged a.a. (Lys, Arg)

Question 15

What is hyperoxaluria?

Answer 15

Build up of calcium oxalate
Kidney stones
AGT enzyme is mistargeted to mitochondria rather than peroxisomes

Question 16

What is the result of a mutation in the glycosylation site in HA?

Answer 16

HA is not glycosylated therefore cannot be folded -> accumulation of unfolded HA in ER