Lecture 13

Question 1

Proteins must be sorted and directed to the _______ and ________ membranes for epithelial polarity to be ______ and ______, and for _______ transport pathways of epithelial cells to function

Answer 1

apical
basolateral
established 
maintained 
ion

Question 2

Proteins, must not only be directed and travel to the correct membrane domain (“traffic”), they must also be ___________ in the membrane correctly

Answer 2

oriented

Question 3

What can affect which membrane the proteins go to?

Answer 3

the polarity

Question 4

How many subunits does ENaC have?

Answer 4

3

Question 5

How many transmembrane domains does each of ENaC’s three subunits have? What else do they have?

Answer 5

2

they also have extracellular loops and intracellular N and C terminals

Question 6

Why is protein trafficking in epithelia important?

Answer 6

because mutations to certain proteins such as CFTR may mean that they can’t get to the membrane or they get there in the wrong orientation, then they will be dysfunctional and the epithelia can’t perform the role that it is supposed to perform

Question 7

Give an example of a mutation in CFTR and what it results in

Answer 7

F508 in the CFTR means that when CFTR reaches the apical membrane, it is not folded properly and so it is not functioning properly this causes cystic fibrosis

Question 8

Give an example of a condition caused by a mutation in ENaC and what this causes

Answer 8

In Liddle’s syndrome, ENaC endocytosis is inhibited causing severe hypertension

Question 9

What do ribosomes do?

Answer 9

They translate mRNA into protein on the ER

Question 10

What happens in the ER?

Answer 10

The proteins can be post-translationally modified and then sent to the golgi

Question 11

What happens in the golgi?

Answer 11

The proteins are further modified and then when they get to the end of the golgi (trans-golgi-network), this sends them off to different parts of the cell wherever they need to go

Question 12

Protein trafficking requires ________ or ’address labels’ in the ________ _________ sequence of a protein

Answer 12

signals

amino acids

Question 13

Plasma membrane proteins may also have a label to ensure they are retrieved from the cell surface by endocytosis, and apical or basolateral signals. True or false?

Answer 13

true

Question 14

Proteins destined for the
‘secretory pathway’ e.g. ER/Golgi/cell surface/lysosomes contain a _________ signal sequence, located at the _______-terminus or further into a protein. ENaC, CFTR and Na+K+ATPase have internal signal sequences in their transmembrane domains.

Answer 14

hydrophobic

N

Question 15

ER localised proteins (ie. proteins that need to go back to the ER) contain the amino acid sequence: KDEL What do these amino acids mean?

Answer 15

(K=lysine, D=aspartic acid, E=glutamic acid, L=leucine).

Question 16

What is the first step in the process of protein trafficking?

Answer 16

synthesis of the protein

Question 17

Describe how the synthesis of a protein works (part 1)

Answer 17

The ribosome will lock on to the mRNA and protein synthesis will begin (at this point, they are not at the ER).
In the first sequence of mRNA that is being translated there is a series of 10-15 hydrophobic amino acids called a signal sequence. This is recognised by a signal recognition particle (which is just floating around looking for the signal sequence). When it finds it, it will bind to it, and it will pull the ribosome with the mRNA and the few amino acids that have been translated, to the ER. As the SRP binds the signal sequence, the synthesis of the protein is inhibited because it has the hydrophobic region so it is taken into the ER.

Question 18

Describe how the synthesis of a protein works (part 2)

Answer 18

It binds to its receptor (SRP receptor) on the ER. This receptor sits close to a pore (a translocon). The ribosome will dock at the ER with the amino acids and there is a resumption of protein synthesis. At this point, the signal sequence (10-15 hydrophobic amino acids), will be cleaved by a signal peptidase enzyme. As it is being chopped off, synthesis is continuing and protein synthesis continues until it is finished. The ribosome dissociates away

Question 19

Where does post-translational modification begins?

Answer 19

in the ER

Question 20

What happens if a protein has a transmembrane domain? (if it only has 1 transmembrane domain)

The ______ is being sequenced by the _______, it is docked etc. and everything up to this point is the same. The _______ enters the __________ via the __________. The __________ will then come across another series of _________ _________. Because they are ______________, they want to stay in the _____________ membrane. This __________ amino acids sequence is called a _______-___________ sequence and this stops the transfer of __________ ___________ and the ___________ into the ______________. This _____________ region will then stay in the _____________, the ___________ will continue synthesis but this time, it is not inside the __________ but the protein is made on the outside of the _________. Synthesis will continue until it is finished. Some of the ___________ is in the ER _________, some is in the __________ and some of it is in the ER ____________.

Answer 20

The mRNA is being sequenced by the ribosome, it is docked etc. and everything up to this point is the same. The protein enters the ribosomes via the translocon. The ribosome will then come across another series of amino acids. Because they are hydrophobic, they want to stay in the ER membrane. This hydrophobic amino acids sequence is called a stop-transfer sequence and this stops the transfer of amino acids and the protein into the ER. This hydrophobic region will then stay in the translocon, the ribosome will continue synthesis but this time, it is not inside the ER but the protein is made on the outside of the ER. Synthesis will continue until it is finished. Some of the protein is in the ER lumen, some is in the cytoplasm and some of it is in the ER membrane.

Question 21

How does a transmembrane domain protein know which place to go?

Answer 21

When a protein is made, anything that is inside the ER will end up outside of the cell (ie. the N terminus). The C-terminus which is in the cytosol will end up still in the cytosol and the transmembrane domain stays in the lipid bilayer

Question 22

What happens if we have 2 transmembrane domains?

Answer 22

There is no signal sequence on the N-terminal end, it is not recognised by the signal recognition particle so it hasn’t been brought to the ER yet. When the ribosome is translating and it gets to the bit destined to be a transmembrane region, this hydrophobic region will represent something similar to that signal sequence, at which point the signal recognition particle will bind to it and the new mRNA and the ribosome will be brought to the ER. It will dock at the ER and the transmembrane domain will be put into the translocon.

Question 23

In something that has two transmembrane domains, the first transmembrane domain is called the what? What is the relevance of this?

Answer 23

start transfer sequence

The ribosome will continue synthesis and these sequence of amino acids will end up in the ER. The ribosome will then come across another hydrophobic region (the second transmembrane domain) which then acts as the stop transfer sequence. The ribosome knows it can continue synthesis but it is going to be outside of the ER

Question 24

What happens if there are more than 2 transmembrane domains?

Answer 24

It follows the same pattern at ENaC and there is a start transfer, a stop transfer, etc until you get passed all the transmembrane domains

Question 25

Which is the correct order for entry of secretory pathway proteins into the endoplasmic reticulum (ER)?
A. Signal peptide cleaved -> protein enters via translocon -> binding of ribosome to ER
B. Protein enters via translocon -> signal peptide cleaved –> SRP (signal recognition particle) binds signal sequence
C. SRP binds signal sequence -> ribosome docks on ER -> protein enters via translocon

Answer 25

C. SRP binds signal sequence -> ribosome docks on ER -> protein enters via translocon

Question 26

What is glycosylation and where does it occur?

Answer 26

This is the adding sugars to proteins and it occurs in the ER

Question 27

What are the two types of glycosylation?

Answer 27

• N-linked

-

Question 28

What is N-linked glycosylation?

Answer 28

When the sugar is added to an asparagine residue in a sequence of NxS/T (S=serine, T = threonine)

Question 29

What is the purpose of glycosylation?

Answer 29

it helps with protein folding

Question 30

As well as glycosylation, what other post-translational modifications happens in the ER?

Answer 30

the addition of GPI anchors

Question 31

What does the GPI do?

Answer 31

it cleaves off the transmembrane domain and the GPI is embedded in the membrane to attach a protein to the membrane
it is a signal for most apical proteins

Question 32

Describe the process of protein folding in the ER

Answer 32

1. there is formation of disulfide bridges between sulfur residues
2. As the SRP binds the signal sequence, the synthesis of the protein is inhibited because it has the hydrophobic region so it is taken into the ER.
3. Assembly of subunits into multiprotein complexes. E.g. ENaC must assemble into a trimer of α-, β- and γENaC subunits. Na+K+ATPase assembles into α plus β subunit complexes

Question 33

What proteins help in folding? Describe how this happens

Answer 33

TRIC

Question 34

Where does folding of proteins and assembly of different protein together occur?

Answer 34

in the ER

Question 35

Describe the ER quality control system

Answer 35

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).
If Na+K+ATPase or ENaC are not assembled or folded correctly they are marked for degradation.

Question 36

Describe how ERAD works

Answer 36

The ribosome is translating mRNA. If the chaperone protein is noticing that the new protein is not being folded correctly, it will tell the ER and the new amino acid sequence is retrotranslocated out of the ER. It it ubiquitinated and removed of glycosylation and the misfolded protein is sent for degredation in the proteasome

Question 37

Describe the mutation causing cystic fibrosis, DF508-CFTR, prevents CFTR reaching the apical membrane and how it is dealt with

Answer 37

Wildtype CFTR protein is made in the endoplasmic reticulum (ER) and is trafficked through the Golgi to the apical membrane where CFTR secretes Cl- or HCO3- ions.
DF508-CFTR is made in the ER but is unable to progress to the Golgi because this protein can’t be folded into the correct structure.
Therefore DF508-CFTR gets stuck in endoplasmic reticulum, can’t move to the Golgi, and instead is tagged by the ubiquitin pathway for destruction in the proteasome (ERAD pathway).

Question 38

Misfolded proteins are targeted for degradation in the lysosome BECAUSE ERAD determines whether proteins are ’ready’ to move further through the secretory pathway

Answer 38

both false

Question 39

Once the protein is made and folded it is packaged in to _______ (called _________) and trafficked to the ________

Answer 39

vesicles
COPII
golgi

Question 40

The Golgi can be further defined as _____, ______ and _______

Answer 40

cis
medial
trans

Question 41

What is the main role of the golgi?

Answer 41

post-translational modifications

Question 42

As well as N-linked glycosylation occurring in the golgi, what other type of glycosylation occurs there? What does this involve?

Answer 42

O-linked which is the addition of sugars to serine or threonines of the proteins

Question 43

What happens at the trans-golgi network

Answer 43

This is a distribution centre - it is here where the proteins are packaged into vesicles and sent where they need to go.

Question 44

What are the two main pathways for how things get from the trans-golgi network to the plasma membrane? Describe them

Answer 44

• constituted pathway: direct route from TGN to membrane
• regulated pathway: the TGN will put the protein into the vesicle, the vesicle sits under the membrane until it is told (eg. by a hormone) to get to the membrane

Question 45

Describe clathrin coated vesicle formation

Answer 45

The protein of interest binds to a receptor in the TGN membrane. These receptors bind to adaptin-clathrin complex which weighs down the membrane and causes it to bud off. When it is fully budded off, the adaptin-clathrin complex falls off and is recycled. The uncoated vesicle can go off were it needs to go

Question 46

Describe clathrin coated vesicle fusion

Answer 46

The clathrin coated vesicles have v-snare proteins which recognise t-snare proteins. The t-snare is on the target membrane and they bind. With the activity of the RAB-GTPase, it allows fusion of the vesicle to the target membrane and you get release of the target membrane into the target cell