Lecture 14

Question 1

Proteins are made in the _____, trafficked to the ______ via _______ vesicles. In the golgi, proteins are __________ _________ __________ until they reach the ________ ________ network. They are then __________ through two different pathways. What are these pathways called and what do they involve?

Answer 1

ER
golgi
COP2
post-translationally modified
trans gogli
exocytosed
constitutive pathway: straight to the plasma membrane or in calthrin coated secretory vesicles via the regulatory pathway

Question 2

What are the basolateral protein sorting signals which tell the protein to go to the basolateral membrane?

Answer 2

tyrosine based motifs (Yxxφ) or dileucine (LL) motifs. But apical proteins also contain these motifs e.g. ENaC has a YXXL, CFTR has YXXφ and LL even though it isn apical protein (we don’t know why)

Question 3

What are the apical sorting signals which tell the protein to the apical membrane?

Answer 3

GPI anchors, O-or N-linked sugar chains, amino acid motifs in the transmembrane domains or cytoplasmic part of the protein.
There is no consensus signal for apical sorting signal, but trafficking to the apical membrane is saturable

Question 4

Trafficking to the apical membrane is saturable which indicates what?

Answer 4

that binding proteins are required to specifically traffic apical proteins

Question 5

The apical sorting signals for CFTR and ENaC are what?

Answer 5

unknown

Question 6

Sorting signals in epithelial cells could depend on heirarchy. What does this mean?

Answer 6

Some transmembrane proteins that have apical and basolateral sorting signals, the basolateral are dominant over the apical signals but ENaC and CFTR have basolateral signals but go to the apical membrane

Question 7

What are the three different routes that proteins could take to get to their respective membranes from the trans-golgi network?

Answer 7

1. direct route
2. indirect route
3. random sorting

Question 8

Describe the direct route of exocytosis

Answer 8

Proteins are sorted at the TGN into either apical or basolateral bound vesicles and are then trafficked straight to their appropriate membrane

Question 9

Describe the indirect route of exocytosis

Answer 9

All new proteins reach the TGN and are then all proteins are exocytosed to one membrane. They are then retrieved from the membrane via endocytosis and trafficked to the endosome. Apical proteins are then trafficked to the apical membrane and basolateral proteins are trafficked to the basolateral membrane.

Question 10

Describe the random sorting route of exocytosis

Answer 10

Proteins in the TGN are trafficked to either membrane randomly. They are then retrieved from the membrane via endocytosis and are trafficked to the endosome. Apical proteins are then redirected to the apical membrane and basolateral proteins are redirected to the basolateral membrane

Question 11

Which route do we think exists most prevalently?

Answer 11

the direct route

Question 12

What is the experimental evidence for the direct route?

Answer 12

• epithelial cells in culture expressing basolateral and apical proteins tagged with different coloured fluorescent tags
• in an experiment, these were cooled to 20 degrees to accumulate proteins at the TGN and were warmed up to 37 degrees and then use live cell imaging to watch the proteins move
• but the cells weren’t fully polarised so there debate about whether there is distinct apical and basolateral membranes and therefore people think that the direct route

Question 13

What is and what is the purpose of an exocyst?

Answer 13

This is another way for the proteins to get to the basolateral membrane. It is a complex of eight proteins important for plasma membrane targeting and tethering of vesicles. It is required for polarised exocytosis in yeast. In polarised epithelial cells ZO proteins connect TJ (in the lateral) to the actin cytoskeleton so basolateral proteins are in exocyst expressing vesicles are then trafficked to the lateral membrane and are then moved down to the basolateral membrane.

Question 14

Which statement about protein sorting and targeting in epithelia is CORRECT?
A. The exocyst is the site for delivery of proteins to the basolateral membrane.
B. Experimental evidence shows that the trans-Golgi network can sort apical and basolateral proteins into separate vesicle populations.
C. In the indirect sorting pathway all proteins are first delivered to the same membrane domain.
D. All of A, B and C are correct.

Answer 14

D. All of A, B and C are correct.

Question 15

Tyrosine motifs are described as Yxxφ. What does this mean?

Give an example

Answer 15

tyrosinse
any two amino acids
a hydrophobic amino acid
eg. ENaC

Question 16

From the TGN, the proteins go to the apical membrane or the basolateral membrane in what?

Answer 16

clathrin coated vesicles

Question 17

Bringing proteins back into the cell is called what?

Answer 17

endocytosis

Question 18

Endocytosis occurs in what?

Answer 18

clathrin coated vesicles (as well as exocytosis)

Question 19

Describe the endocytosis of the proteins in the membrane via clathrin coated vesicles

There are __________ in the membrane which recruit ___________ and ___________ and you get an _________/__________ complex. _________ will bind to the receptor which causes a ________ down of the _________ and causes it to ___________ in. This is called _________ __________ endocytosis.
There is another form of clathrin coated endocytosis where they do not bind receptors and it is just a __________ uptake of _________. This is called ___________ phase endocytosis.
The __________ buds off from the ___________ (containing the endocytosed material). The ___________ carries this material away from the __________ and once it gets far enough away from the membrane, the __________ and __________ dissociate and they are recycled back to the __________ in the ___________. The uncoated ____________ vesicle approaches the ______________. The ___________ on the vesicle connects with the ____________ on the target membrane (in this case, the ____________ membrane). There is then a fusion of the ________ with the ___________ and the vesicle empties its contents into the __________.

Answer 19

There are receptors in the membrane which recruit adaptin and clathrin and you get an adpatin clathrin complex. Proteins will bind to the receptor which causes a weighing down of the membrane and causes it to bud in. This is called receptor mediated endocytosis.
There is another form of clathrin coated endocytosis where they do not bind receptors and it is just a random uptake of ECF. This is called fluid phase endocytosis.
The vesicle buds off from the membrane (containing the endocytosed material). The vesicle carries this material away from the membrane and once it gets far enough away from the membrane, the clathrin and adaptin dissociate and they are recycled back to the receptors in the membrane. The uncoated clathrin vesicle approaches the endosome. The v-SNARE on the vesicle connects with the t-SNARE on the target membrane (in this case, the endosomal membrane). There is then a fusion of the vesicle with the membrane and the vesicle empties its contents into the endosome.

Question 20

As well as clathrin coated endocytosis, there is another form. What is this called?

Answer 20

This is called calveoli-mediated endocytosis.

Question 21

Describe calveoli-mediated endocytosis
Give an example of when this is useful

There are small ________-raft ___________ of the membrane and a small protein called ____________ associates with the ________ to form _________-coated vesicle. _______ bind to the receptors and _________ bud off from the _________. These then fuse with a ___________ which is a type of early __________ but once the _____________ vesicle fuses with the __________, the _________ is then able to fuse with other __________ compartments, the __________, or even go back to the ________ membrane.
This is useful in the trafficking of __________ which maintains the fluidity of the plasma membrane.

Answer 21

There are small lipid-raft invaginations of the membrane and a small protein called calveolin associates with the rafts to form calveolae-coated vesicle. Ligands bind to the receptors and calveolae bud off from the membrane. These then fuse with a caveosome which is a type of early endosome but once the calveolae-coated vesicle fuses with the caveosome, the caveosome is then able to fuse with other endosomal compartments, the lysosome, or even go back to the plasma membrane.
This is useful in the trafficking of cholesterol which maintains the fluidity of the plasma membrane.

Question 22

How do our cells know what and what not to endocytose?

Give three examples

Answer 22

There are signals such as tyrosine based motifs such as Yxxφ or NPXY or hydrophobic amino acid motifs such as dileucine (LL) or monoubiquitination
ENaC subunits have YXXφ motifs and are monoubiquitinated, CFTR has YXXφ and LL motifs and Na+/K+ATPase subunits have YXXφ and LL motifs.

Question 23

Endocytosis motifs are bound by what?

Answer 23

clathrin-binding adaptins (receptors of clathrin coated vesicles)

Question 24

What is a name for a specific adaptin which is present in the clathrin coated vesicles?

Answer 24

AP2

Question 25

How many subunits is AP2?

What are they and what do they do?

Answer 25

4

α and β and they bind to clathrin, μ2 binds to the Yxxφ motif and σ2 subunit

Question 26

Endocytosis of transmembrane proteins occurs through interaction with the AP2 complex BECAUSE the transmembrane proteins contain amino acid sequences such as YXXφ.
A. Both statements true and causal.
B. Both statements true but not causal.
C. First statement true, second false.
D. First statement false, second true.
E. Both statements false

Answer 26

both statements are true and causal

Question 27

Describe endocytosis in endothelial cells

Answer 27

1a. Apical proteins are endocytosed to an apical sorting endosome
1b. Basolateral proteins are endocytosed to a basolateral sorting endosome
2. Endocytosed material (apical and basolateral) goes to the common endosome
3a. Basolateral proteins can be recycled back to the basolateral membrane
3b. Apical proteins are, believed, to recycle back to the apical membrane via an apical recycling endosome

Question 28

Describe the endocytosis and Recycling or degradation of apical ENaC

Answer 28

ENaC is ubiquitinated and endocytosed
It is Trafficked through the apical sorting endosome to the common endosome and then if it is going to be recycled, the Ubiquitin is removed- recycled back to apical membrane via the apical recycling endosome.
If it is going to be degraded Ubiquitin remains– trafficked to late endosome and onto the lysosome for degradation

Question 29

Endocytosis and recycling or degradation of the basolateral transferrin receptor (TfR)

Answer 29

1. Once ligand-bound transferrin is endocytosed to basolateral sorting membrane
2. Trafficked on to common endosome
3. TfR is either recycled back to basolateral membrane or trafficked through the late endosome to the lysosome for degradation

Question 30

Describe the transcytosis of the basolateral protein immunoglobulin A receptor - IgAR

Answer 30

1. IgAR endocystosed from basolateral membrane and trafficked to basolateral sorting endosome
   2 & 3. Trafficked on to apical recycling endosome through common endosome
2. IgAR exocytosed to apical membrane