Lecture 13 - protein trafficking in epithelial cells 2

Question 1

why is protein trafficking in epithelia important?

Answer 1

genetic polymorphisms or mutations alter protein trafficking pathways to reduce or increase cell surface population

eg. prevent DF508-CFTR reaching the apical membrane

CFTR is trafficked to the apical membrane to secrete chlrode ions.

DF508-CFTR gets stuck in endoplasmic reticulum and is degraded.

Question 2

Pathways of protein trafficking in the cell

Answer 2

secretion

1. from peroxisome, nucleus, mitochrondria
2. cytosol
3. endoplasmic reticulum
4. golgi
5. secretory vesicles, lysosome, multivesicular body, early/ sorting endosome
6. cell surface

Question 3

why do protein trafficking requires signals in the amino acis squence of a protein?

Answer 3

proteins contain address labels to interact with cellular pathways to ensure they are localised correctly in the cell. Plasma membrane proteins may also have a label to ensure they are retrieved from the cell surface by endocytosis and apical or basolateral signals

Question 4

hydrophobic signal sequence

Answer 4

Proteins destined for secretory pathways eg, ER/Golgi/ cell surface/lysosomes contain this. it is located at the N-terminus or futher into a protein. ENaC, CFTR and NA/KATPase have internal signal squence in their transmembrane domains.

Question 5

what amino acis squence does the ER localised proteins contain?

Answer 5

KDEL

• K=lysine
• D=aspartic acid
• E=glutamic acid
• L=leucine

Question 6

entry of protein into ER - example of a soluble protein that will be secreted from the cell

Answer 6

Question 7

what is the correct order for entry of secretory pathway proteins into endoplasmic reticulum?

Answer 7

SRP binds signal sequence - ribisome docks on ER - protein enters via translocon

Question 8

entry of proteins into ER - example of a transmembrane protein that will be embedded in plasma membrane

Answer 8

Question 9

what do the hydrophobic and postive stretches of amino acids do?

Answer 9

they set up the topology of membrane proteins with two transmembrane domains

ENaC subunits have 2 transmembrane domains, N and C termini are cytosolic

NA/KATPase alpha subunit has 10 transmembrane domains

Question 10

post translational modifications in the ER - glycosylation/ addition of sugars

Answer 10

• plasma menbrane proteins are often glycosylated (sugars added) on asparagines in the amino acid sequence.
• ENaC, CFTR and Na/K/ATPase are all gycosylated

Question 11

what do the chaperones calnexin and calreticulum do?

Answer 11

they bind to glycosylated CFTR and help with folding CFTR

Question 12

post translational modifications in the ER - GPI anchors

Answer 12

• glycosylphosphatidylinositol (GPI) Anchors may replace transmembrane domains
• GPI anchors are usally found on apical proteins

Question 13

folding and assembly

Answer 13

1. disulfide bridges/bonds formed in alpha, beta and gamma subunitsd of ENaC
2. Correct folding must occur, mediated by molecular chaperone proteins
3. assembly of subunits into multiprotein complexes

Question 14

protein folding

Answer 14

1. unfolded protein enters TRIC
2. protein folds inside of TRIC
3. folded protein exts the TRIC

Question 15

ER Quality Control system

Answer 15

• ER recognises whether proteins are ‘ready’ to move onto the golgi, need to stay in the ER longer, or whether they need to be destroyed.
• chaperones determine if a protein is incorrectly folded or misassembled. these proteins are sent for degradation (ERAD)
• cystic fibrosis causes retention of CFTR in ER

Question 16

ERAD

Answer 16

1. Substrate recognition
2. Retrotranslocation
3. Ubiquitination
4. Deglycosylation
5. degradation in proteasome

Question 17

transport to the golgi network via vesicles

Answer 17

1. golgi to ER (COPI vesicles)
2. ER to golgi (COPII vesicles)
3. golgi to cell surface (clathrin vesicles)

Question 18

post translational processing in the GOLGI

Answer 18

• modification of sugars/ N-linked glycosylation occurs
• addition of sugars to serine/threonines of proteins = O-linked glycosylation
• sulfation of sugars and some tyrosines

Question 19

prtoein sorting at the trans-golgi network

Answer 19

ENac and Na/K/ATPase use the constitutive pathway

Question 20

clathrin- coated vesicle formation and fusion

Answer 20

• clathrin coated vesicles move proteins from the gogi to the plasma membrane.
• v-share protein binds to t-share protein on target membrane and with Rab they force vesicle fusion with cell membrane.