lecture 13

Question 1

describe ENaC

Answer 1

it has 3 subunits (alpha, beta, gamma) and 3 large extracellular loops with a C and an N terminus

each subunit has 2 transmembrane domains

Question 2

what are some reasons that cause alterations in protein trafficing pathways

what are some diseases that happen when things go wrong

Answer 2

Genetic polymorphisms or mutations alter protein trafficking pathways to reduce or increase cell surface populations disrupting ion transport

pathways e.g. some examples of this that lead to diseases are….

Very little DF508-CFTR reaches the apical membrane causing cystic fibrosis. this happens because the protein is folded incorrectly leading to degradation within the cell

In Liddle’s syndrome ENaC endocytosis is inhibited causing severe hypertension (this mutation in on intracellular parts).

Question 3

what causes cystic fibrosis

Answer 3

Very little DF508-CFTR reaches the apical membrane causing cystic fibrosis. this happens because the protein is folded incorrectly leading to degradation within the cell

Question 4

what is Liddle’s syndrome

Answer 4

In Liddle’s syndrome ENaC endocytosis is inhibited causing severe hypertension (this mutation in on intracellular parts).

Question 5

what is the general pathway of protein trafficking

Answer 5

Ribosomes translate mRNA into proteins

It then gets packaged and sent to the Golgi where it is modified and packaged into vesicles and sent to where the are supposed to be

Trans Golgi network is what tells the vesicles where to go

Question 6

when would proteins have a hydrophobic signal sequence

Answer 6

when they are destined for a secretory pathway

eg they are destined for the ER, Golgi, cell surface

Question 7

where would the hydrophobic signal sequence be located in secretory pathway proteins

Answer 7

at the N terminus or further into the protein

Question 8

where do ENaC, CFTR and Na/K ATPase have there signal sequence

Answer 8

they have internal signal sequences in their transmembrane domains

Question 9

ER locolised protein have what amino acid sequence

Answer 9

KDEL

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

KDEL tels the golgi to put the proteins back into the ER

Question 10

what acts as an address as to where the proteins are supposed to go

Answer 10

the amino acid sequence

Question 11

which is the first part of the protein to be translated

Answer 11

the N terminus

the hydrophobic region is usually in close proximity to this

Question 12

describe how protein synthesis works for a simple protein (no transmembrane domains)

Answer 12

The ribosome locks onto some mRNA and protein synthesis will begin. At this point they are not in the Endoplasmic reticulum

There is a series of 10 - 15 hydrophobic amino acids which is the signal sequence. This will be recognised by a signal recognition particle which is floating around looking for the sequence

When it finds it, it will pull the mRNA along with the ribosome and the few amino acids that have been translated to the ER. When it brings it there, it will bind to the signal recognition particle receptor (SRPR) which is located very close to a translocon (Brown thing) which is located very close to a pore

When it binds the sequence it inhibits protein synthesis because it is bound to the hydrophobic region and in in the cytoplasm therefore it is not happy. therefore it has to be inside the ER before synthesis will resume (attached to the membrane receptor translocon).

the hydrophobic sequence (signal sequence) is cleaved by an enzyme called signal peptidase. Once the hydrophobic region is chopped off protein synthesis can be completed

At this point (after synthesis is compleated) the ribosome is able to disassociate away and the protein is inside the ER.

There is also an addition of carbs as in the ER sugars can be added onto proteins as the ER is where posttranslational modification begins

Question 13

does the ribosome bind inside or outside of the ER

Answer 13

outside

Question 14

what happens initially when the ribosome binds

Answer 14

it translates a series of 10-15 hydrophobic amino acids which is the signal sequence

Question 15

what pulls the mRNA with the ribosome and the few amino acids that have been translated to the ER

Answer 15

the signal recognition particle

Question 16

what receptor does the signal recognition particle bind

Answer 16

the signal recognition particle receptor

Question 17

what is in close proximity to the signal recognition particle receptor

Answer 17

a translocon (pore)

Question 18

what happens when the hydrophobic region is inside the ER attached to the receptor

Answer 18

signal peptidase cleaves off the signal sequence

allowing for the rest of the sequences to be translated

Question 19

what sets up the orientation (topology) of membrane proteins

Answer 19

hydrophobic and positive stretches of amino acids

Question 20

what is the difference in synthesis between a simple protein and a protein with 1 transmembrane domain

Answer 20

after signal peptidase cleaves of the signal sequence and translation starts again, it will come across another hydrophobic region that will want to stay in the membrane

This is called a stop transfer sequence. The stop transfer sequence will stay in the translocon and the rest of the protein will be made outside of the ER

When synthesis is compleart the ribosome will dissocosite. Outside the ER is the C terminal and inside in the N

Things made in the ER will end up outside the cell, cytosol part will end up inside the cell and the transmembrane is in the transmembrane

Question 21

describe synthesis for 2 transmembrane domains

Answer 21

There is no signal sequence on the N terminal end therefore it is not recognised by the signal recognition pathway and they ribosome will start making the protein outside of the ER

the ribosome will eventually come to a hydrophobic region that resembles the start sequence. This is when the recognition particle will bind to it and pull it to the translocon.

The first transmembrane domain acts as the signal sequence (but it is not cleaved by signal peptidase).

The first transmembrane domain is called the start transfer sequence

When it comes across another hydrophobic sequence this is the second transmembrane domain which is the stop sequence

Question 22

what is the start transfer sequence

Answer 22

it is the first transmembrane domain of a protein with 2 or more transmembrane domains

this acts as a signal sequence (but doesn’t get cleaved off by signal peptidase)

Question 23

what is the stop transfer sequence

Answer 23

it is every 2nd transmembrane domain of a protein with 2 or more transmembrane domains

it is the stop signal

Question 24

in ENaC where are the N and C terminals located

Answer 24

N and C terminals are cytosolic (inside the cell)

Question 25

the alpha subunit in Na/K ATPase has how many transmembrane domains

Answer 25

10

Question 26

CFTR has how many transmembrane domains

Answer 26

12

Question 27

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 27

C

Question 28

what proteins are often glycosylated

Answer 28

plasma membrane proteins often have sugar groups added

Question 29

what does glycosylation help with

Answer 29

protein folding

Question 30

what does NxS/T stand for (N-linked glycosylation)

Answer 30

N-linked glycosylation is where a sugar is added to the asparagine residue N=asparagine, x=any amino acid, S=serine, T=threonine

Question 31

where are GPI anchors usually found

Answer 31

on apical membranes

Question 32

what can GPI anchors replace

Answer 32

transmembrane domains

Question 33

how can GPI anchors replace transmembrane domains

Answer 33

it is a glycophospholipid which is found in the membrane. when it cleaves off the transmembrane domain (through phosphorylation) the GPI is covalently bonded to the protein therefore anchoring it to the membrane

Question 34

what is the first thing to happen in terms of protein folding

Answer 34

disulfide bridges form

Question 35

what is folding mediated by

Answer 35

molecular chaperone proteins

Question 36

what is the ERs quolity control system

Answer 36

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 37

what degrades protein which have been incorrectly folded/misassembled

Answer 37

ERAD ER assisted degradation

Question 38

describe the process of ERAD

Answer 38

if the chaperone notices something is going wrong then the amino acid sequence will be reterolocated out of the ER from here it will be Ubiquitinated and the sugars will be removed the amino acid sequence will then be degraded in a proteasome

Question 39

the most common mutation in CFTR prevents what

Answer 39

prevents CFTR from reaching the apical membrane

Question 40

what happens that prevents CFTR from reaching the apical membrane

Answer 40

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 41

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

Answer 41

first statement should say proteasome second statment is false because it is the ER not ERAD that determines it therefore both statements are false

Question 42

what vesicles take correctly folded proteins from the ER to the golgi

Answer 42

COP2

Question 43

what are the 3 parts of the golgi called

Answer 43

cis, medial and trans

Question 44

what is the role of the golgi

Answer 44

posttranslational modifications

Question 45

what are the 3 post translational modifications the golgi does

Answer 45

N linked glycosylation Addition of sugars to serine/threonines of proteins = O-linked glycosylation. Sulfation of sugars and some tyrosines.

Question 46

what kinds of post translation modifications does ENaC undergo

Answer 46

``` ENaC is extensively post-translationally modified and proteolytically cleaved ``` it undergoes both N and O linked glycosylation

Question 47

ENaC, CFTR and Na+K+ATPase complexes move to plasma membrane via what pathway (what is special about ENaC)

Answer 47

constitutive pathway (direct pathway) HOWEVER Also evidence for ENaC (and AQP2) using the regulated pathway – proteins stored in vesicles under apical membrane until signal induces exocytosis. (regulated pathway)

Question 48

what is the role of the trans Golgi network

Answer 48

it is the distribution centre this is where proteins are packaged into vesicles and sent to where they need to go

Question 49

what kind of vesicles move proteins from the golgi to the plasma membrane (what are they coated in)

Answer 49

clatheran coated vesicles

Question 50

how do vesicles form on the trans Golgi membrane

Answer 50

you will have specific protein receptors on the inside of the trans golgi membrane that will bind specific proteins which will weigh down the membrane. one the protein is bound to its receptors the adaptin clathrin complex will bind to the outside of the trans golgi membrane further weighing it down causing tha part of the membrane to budd off you now have a clathrin coated vesicle

Question 51

how do vesicles get exocytosed into the cell membrane

Answer 51

when the vesical buds off from the trans Golgi membrane the clathrin adaptin complex comes off which allows the vesicles to fuse easily with the membrane v-SNARE protein on vesicle binds t-SNARE protein on target membrane. With Rab they force vesicle fusion with cell membrane.