Lecture 13 Flashcards
(46 cards)
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
apical basolateral established maintained ion
Proteins, must not only be directed and travel to the correct membrane domain (“traffic”), they must also be ___________ in the membrane correctly
oriented
What can affect which membrane the proteins go to?
the polarity
How many subunits does ENaC have?
3
How many transmembrane domains does each of ENaC’s three subunits have? What else do they have?
2
they also have extracellular loops and intracellular N and C terminals
Why is protein trafficking in epithelia important?
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
Give an example of a mutation in CFTR and what it results in
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
Give an example of a condition caused by a mutation in ENaC and what this causes
In Liddle’s syndrome, ENaC endocytosis is inhibited causing severe hypertension
What do ribosomes do?
They translate mRNA into protein on the ER
What happens in the ER?
The proteins can be post-translationally modified and then sent to the golgi
What happens in the golgi?
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
Protein trafficking requires ________ or ’address labels’ in the ________ _________ sequence of a protein
signals
amino acids
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?
true
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.
hydrophobic
N
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?
(K=lysine, D=aspartic acid, E=glutamic acid, L=leucine).
What is the first step in the process of protein trafficking?
synthesis of the protein
Describe how the synthesis of a protein works (part 1)
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.
Describe how the synthesis of a protein works (part 2)
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
Where does post-translational modification begins?
in the ER
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 ____________.
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.
How does a transmembrane domain protein know which place to go?
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
What happens if we have 2 transmembrane domains?
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.
In something that has two transmembrane domains, the first transmembrane domain is called the what? What is the relevance of this?
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
What happens if there are more than 2 transmembrane domains?
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
