JC1

Question 1

what is the most prevalent cystic fibrosis causing mutation?

Answer 1

deletion of a phenylalanine at position 508 in CFTR

Question 2

how does Δf508 CFTR effect traffiking of CFTR?

Answer 2

defects in golgi-mediated exocytosis and reduction in/no cell surface expression

Question 3

How can cell surface expression of CFTR be rescued in Δf508 mutant?

Answer 3

-directing Δf508 CFTR via the unconventional GRASP-dependent secretory pathway

Question 4

What causes cell surface traffiking to use unconventional GRASP-dependent route?

Answer 4

ER stress

- induces cell surface trafficking of mutant and WT CFTR via GRASP-dependent pathway

Question 5

what domains/interactions are critical for unconventional cell surface trafficking?

Answer 5

• Phosphoylations of a specific site of GRASP

- PDZ-based interaction between GRASP55 and CFTR

Question 6

what are band C and band B CFTR?

Answer 6

band c - fully glycosylated mature CFTR (WT)

band b - ER-core glycosylated immature form (mutant)

Question 7

what usually happens to ΔF508 CFTR at the ER?

Answer 7

• Due to deleted phenylalanine there is misfolding

- this causes retention in the ER (undergoes rounds of CNX cycle) and eventual degradation via ERAD

Question 8

what does GRASP stand for?

Answer 8

golgi reassembly stacking proteins

-involved in unconventional secretion of proteins which bypass the golgi

Question 9

what are the TM sensor proteins in the ER that detect ER stress?

Answer 9

activated when abundance of unfolded nascent polypeptide chains in the ER lumen

• PERK
• IRE1
• AFT6

(mediate/activate UPR)

Question 10

How was blocking of ER-golgi transport acheived (multiple methods)?

Answer 10

1) dominant negative SAR1 (component of COPII complex)
2) overexpression of STX5 - disrupt balance of SNARE complexes
3) dominant negative Arf (component of COPII)

Question 11

Is immature core glycosylated CFTR functional at the cell membrane if it is able to reach the membrane?

Answer 11

YES - is functional at the membrane

-shown as produces Cl- current

Question 12

how can ER stress be induced?

Answer 12

• brefeldin blocked ER-golgi traffiking - accumulation of secretory proteins in ER -UPR
• thapsigargin - depletes calcium stores - accumulation of vesicles in ER - UPS

Question 13

How can ER stress be measured?

Answer 13

production of ER stress marker protein - CHOP

Question 14

what does transgenic expression of GRASP in mice do?

Answer 14

• it rescues ΔF508 mouse survival
• mice survive longer than without GRASP55(WT mice dont need GRASP 55 - shows not just GRASP55 but its interaciton with CFTR is important)
• reverses growth retardation
• current measurments more normal