lecture 13 Flashcards
describe ENaC
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
what are some reasons that cause alterations in protein trafficing pathways
what are some diseases that happen when things go wrong
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).
what causes cystic fibrosis
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
what is Liddle’s syndrome
In Liddle’s syndrome ENaC endocytosis is inhibited causing severe hypertension (this mutation in on intracellular parts).
what is the general pathway of protein trafficking
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
when would proteins have a hydrophobic signal sequence
when they are destined for a secretory pathway
eg they are destined for the ER, Golgi, cell surface
where would the hydrophobic signal sequence be located in secretory pathway proteins
at the N terminus or further into the protein
where do ENaC, CFTR and Na/K ATPase have there signal sequence
they have internal signal sequences in their transmembrane domains
ER locolised protein have what amino acid sequence
KDEL
(K=lysine, D=aspartic acid, E=glutamic acid, L=leucine).
KDEL tels the golgi to put the proteins back into the ER
what acts as an address as to where the proteins are supposed to go
the amino acid sequence
which is the first part of the protein to be translated
the N terminus
the hydrophobic region is usually in close proximity to this
describe how protein synthesis works for a simple protein (no transmembrane domains)
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
does the ribosome bind inside or outside of the ER
outside
what happens initially when the ribosome binds
it translates a series of 10-15 hydrophobic amino acids which is the signal sequence
what pulls the mRNA with the ribosome and the few amino acids that have been translated to the ER
the signal recognition particle
what receptor does the signal recognition particle bind
the signal recognition particle receptor
what is in close proximity to the signal recognition particle receptor
a translocon (pore)
what happens when the hydrophobic region is inside the ER attached to the receptor
signal peptidase cleaves off the signal sequence
allowing for the rest of the sequences to be translated
what sets up the orientation (topology) of membrane proteins
hydrophobic and positive stretches of amino acids
what is the difference in synthesis between a simple protein and a protein with 1 transmembrane domain
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
describe synthesis for 2 transmembrane domains
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
what is the start transfer sequence
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)
what is the stop transfer sequence
it is every 2nd transmembrane domain of a protein with 2 or more transmembrane domains
it is the stop signal
in ENaC where are the N and C terminals located
N and C terminals are cytosolic (inside the cell)
the alpha subunit in Na/K ATPase has how many transmembrane domains
10
CFTR has how many transmembrane domains
12
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
C
what proteins are often glycosylated
plasma membrane proteins often have sugar groups added
what does glycosylation help with
protein folding
what does NxS/T stand for (N-linked glycosylation)
N-linked glycosylation is where a sugar is added to the asparagine residue
N=asparagine,
x=any amino
acid,
S=serine,
T=threonine
where are GPI anchors usually found
on apical membranes
what can GPI anchors replace
transmembrane domains
how can GPI anchors replace transmembrane domains
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
what is the first thing to happen in terms of protein folding
disulfide bridges form
what is folding mediated by
molecular chaperone proteins
what is the ERs quolity control system
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
what degrades protein which have been incorrectly folded/misassembled
ERAD
ER assisted degradation
describe the process of ERAD
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
the most common mutation in CFTR prevents what
prevents CFTR from reaching the apical membrane
what happens that prevents CFTR from reaching the apical membrane
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).
Misfolded proteins are targeted for degradation in the lysosome
BECAUSE
ERAD determines whether proteins are ’ready’ to move
further through the secretory pathway
first statement should say proteasome
second statment is false because it is the ER not ERAD that determines it
therefore both statements are false
what vesicles take correctly folded proteins from the ER to the golgi
COP2
what are the 3 parts of the golgi called
cis, medial and trans
what is the role of the golgi
posttranslational modifications
what are the 3 post translational modifications the golgi does
N linked glycosylation
Addition of sugars to
serine/threonines of proteins = O-linked glycosylation.
Sulfation of sugars and some tyrosines.
what kinds of post translation modifications does ENaC undergo
ENaC is extensively post-translationally modified and proteolytically cleaved
it undergoes both N and O linked glycosylation
ENaC, CFTR and
Na+K+ATPase complexes
move to plasma
membrane via what pathway
(what is special about ENaC)
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)
what is the role of the trans Golgi network
it is the distribution centre
this is where proteins are packaged into vesicles and sent to where they need to go
what kind of vesicles move proteins from the golgi to the plasma membrane (what are they coated in)
clatheran coated vesicles
how do vesicles form on the trans Golgi membrane
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
how do vesicles get exocytosed into the cell membrane
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.
