Intracellular Trafficking Lectures Flashcards
What is the function of Cystic Fibrosis Transmembrane Regulator (CFTR)? What happens if there is a mutation?
It is a chloride channel that maintains hydration in lung airways
Mutation causes loss of CFTR function and Cystic Fibrosis
Involves ER translocation, chaperon-assisted folding, ERAD
What is the first hereditary disease to have the gene identified and sequenced?
Cystic Fibrosis Transmembrane Regulator
Describe the CTFR sequence.
- 1480 amino acids
- 12 TM helices
- 2 cytosolic nucleotide-binding domains (NDB)
- cytosolic N-terminus and loops
Describe translocation in CTFR sequence. (translocation; A genetic change in which a piece of one chromosome breaks off and attaches to another chromosome)
TM1 and TM7 act as Signal Anchors
charged around TM1 and TM7 determine orientation in membrane
other TM helices are threaded in and out
N-linked glycan attached during translocation
If there is a deletion of Phe in NBD1 in CTFR protein, what happens?
This is the most common disease mutation!
The mutation disrupts the hydrophobic core of NBD1 and therefore its folding.
It cannot interact correctly with TM helices or NBD2, so then it is retained in the ER and then degraded, instead of trafficking to the plasma membrane.
What is the function of CFTR ERAD? How does it work?
- ERAD is a major quality control pathway of the cell (endoplasmic reticulum-associated degradation) - the unfolded proteins are recognized by this system!
- If there is a misfolded mutant, CFTR is selected for ERAD by E3 ligases. These complexes are in the ER.
- There are also specialized co-chaperones that promote ERAD
- p97 helps to extract mutant CFTR from membrane
- HS70- CHIP complexes in cytosol, aids in ubiquitination-degredaiton
What is the function of the secretory pathway?
Proteins are properly folded in the ER and they need to go to the plasma membrane, and this journey is done by vesicles.
What is coated vesicle transport? And what are the three types?
- Coated vesicles have a critical role in concentrating, packaging, and shuttling cargo between different intracellular compartments and plasma membrane domains
- COP-II vesicles transport from ER to Golgi (anterograde)
- COP-I vesicles transport from Golgi back to ER (retrograde)
Both have related mechanism but different proteins involves - Clathrin-coated vesicles (CCV) transport from Golgi and PM to endosomes
What are the 4 common parts to all vesicle formation?
- Initiation (some event on the membrane starts the process of forming a vesicle)
- Coat formation (vesicles needs to be coated to then get punch -
A. cytosolic adaptor proteins interact with initiator
B. adaptors collect transmembrane cargo, or cargo receptors (bring coat of vesicle)
C. coat: protein framework is formed on top of adaptors to shape the vesicle bud from the membrane - Fission
A. bud is pinched off to separate the vesicle from membrane - Uncoating
A. coat is removed to allow vesicle targeting and fusion
What is the function of the Ras GTPase Family of Proteins?
Describe the different elements.
They bind GTP and induce a conformational change in the protein. When the protein has the conformation, the protein is then active, they can now bind various effectors. Once is is active, it provides a binding site! There is a cycle to make it active.
When it is GDP, it is in a Inactive monomeric GTPase. It meets with GEF and releases GDP and then forms GTP which is the active GTP monomeric GTPase, and then it meets GAP which makes it back inactive (this is cycle).
Describe COP-II Vesicle Initiation:
COP-II vesicles are for specific ER exit sites
Proteins with exit signals are collected (cargo receptors)
Misfolded proteins are kept away (calnexin)
Sar1-GTP exposes amphipathetic helix (hidden inside the protein, it is polar and non-polar region, in this form it is not active) and it partially insets into the membrane, which then initiates vesicle formation. GDP is then released and GTP enters and there is now an acitve-membrane bound Sar1-GTP.
How is the coat of COP-II formed?
They recruit adaptors. There are adaptor proteins (Sec23 and Sec24) which bind activated Sar1 and TM cargo proteins, or cargo receptors for lumenal proteins. Then there are coat proteins (Sec13 and Sec24) that bind adaptors and shape membrane into vesicles. Now that the coat is completed, it pinches the vesicle off the membrane. The energy for this shaping and pinching off the vesicle is only from protein interactions, it is not from GTP hydrolysis. Therefore for this to work, you only need the interactions of the proteins.
What is the process of COP-II Uncoating? Why is this step necessary?
It is necessary for vesicle fusion at the Golgi.
The coat (Sec12/31) forms a cage-like structure around the vesicle. And there are Adaptors (Sec23/24) that act as GAP that allow Sar1 to hydrolyze GTP. But then it needs to be uncoated!
How are proteins exported from the ER?
Bulk flow!
-Proteins in the ER are transported to the Golgi and PM by default, even with no exit signals. But many proteins are exported much more efficiently, while some other returns to ER.
What proteins have exit signals in the ER?
- TM proteins have exit signals on the cytosolic side
- Lumenal proteins are recognized by various cargo receptors, which are TM proteins with exit signals (FF)
What is the ER Retrieval pathway?
In the retrival pathway, proteins are packaged into COPII vesicles and transported to the Golgi, they are then retrieved via the retrograde transport pathway. In the ER, substrates of both pathways converge for ERAD.
After the ER resident proteins are transported to Golgi by bulk flow, they have signals that return them to the ER.
What are the two types of proteins involved in ER Retrival?
- Lumenal proteins
- KDEL-COO- at C-terminus
- recognized by transmembrane KDEL receptor, which itself has a KKxx motif at the cytosolic C-terminus - TM proteins
- KKxx-COO- at cytosolic C-terminus
- NH2+= MxxRR at cytosolic N-terminus
- motifs are recognized by the COP-I coat adaptors
How do COP-I coated vesicles function?
- ArF1- GTP initiator insets amphipathic helix into membrane
- Adaptors and Coats are unrelated to COP-II but function similarly
- Adaptors are GAPS for Arf1, to dissociate coat
Describe the function of Clathrin-coated Vesicles (CCV) and PI:
PI-phosphates in PM and Golgi initiate vesicle formation. PI on cytosolic face of membranes can be phosphorylated at different hydroxyl positions by PI kinases. It provides binding sites for different proteins.
PI(4,5)P2: PM clathrin adaptors, dyamin is the main differentce between the two types of vesicles, signal for formation of adaptors
PI(4)P: Golgi clathrin adaptors
They are unique because they have sugar in the polar head, they are rare but important for the activation of kinase and cascades.
What adaptor proteins are associated with CCV? (Clathrin-Coated Vesicles)
Adaptor proteins (AP-1, AP-2, and some others) are what bind PI-phosphates and cargo in the membrane. There are many different signals for the selection of cargo, include mono-ubuquitination, and phosphorylation.
The Arf GTPase assist some adaptors but they do not do any of the initiation.
Describe the Clathrin Cage (what shape does it form etc.) And its function.
It forms a triskelion shape. It is made up of oligomers and 3 heavy + 3 light chains. They assemble on adaptors to shape the membrane and then form “coated pits.” Once the cage is formed around the vesicle (it is bigger than the other two cages) it cannot pinch because it needs an extra protein. This protein is Dynamin (GTPase protein). This is what makes it different.
Describe the Fission step of CCV.
Dynamin GTPase pinches off CCVs - not a member of Ras family.
The dynamin monomers assemble in TP-bound state into oligomeric rings at the base of the bud. The GTP hydrolysis causes a coordinated constriction of ring that pinches off the vesicle!
The rings then disassemble in GDP-bound state.
Describe the process of Vesicle Traffic.
Membrane vesicle trafficking (also called cell trafficking) is the transport of cell products between subcellular components like the Endoplasmic Reticulum (ER) and Golgi apparatus via membrane encapsulated vesicles, to and from the plasma membrane and other specific cell locations.
There are multiple donor and acceptor (target) membranes in the secretory pathway. Two mechanisms ensure that vesicles transport their contents to the correct acceptor membrane.
1) Rab GTPase proteins provide specificity of vesicle targeting and attachment to acceptor membrane (traffic 2)
2) SNARE fusion proteins provide specificity during fusion of vesicles with acceptor membrane (traffic 3)
Why are there multiple steps in the secretory pathway?
The Golgi apparatus
How is the golgi organized?
It is organized into a stack of membranes.
Cis, medial, and trans membrane.
Compare the old model of transport through the golgi to the new model:
The first model assumed that all membranes of the Golgi is static (they are always the same). The vesicles are the only thing that move (from one to another).
Model two assumes that each layer matures and becomes the next. The movement of systems from cis to trans - it is not static. - Golgi-resident proteins are carried backwards by COP-I vesicles. Clathrin-coated vesicles carry cargo to PM and edosomes.
What maintains the organization of the stack in the Golgi?
A cytosolic protein matrix
What is the process of Glycosylation in the Golgi?
N-linked glycans are modified by removal or mannoses and there is addition of different sugars, often with a negative charge. Other complex oligosaccharides are attached to Ser and Thr side chains - O- linked glycosylation.
There are many different combination that could take place - heterogeneity.
This only happens in the Golgi and you need specific amino acids.
What is the function of glycosylation in the Golgi?
This promotes protein folding
Makes protein folding intermediates more soluble (prevents aggregation)
The modification happens in sequences.
The sugars have limited flexibility, so they protect from proteases and stabilizes protein structure (protein coat)
There are signalling hubs that regulate development. The main function is to protect because we don’t want the proteins to be degraded.
Describe the modification in the Golgi: Proprotein Convertases: Provide two examples.
Some plasma membrane and extracellular proteins are made as longer, inactive form at the ER and then they are cut by proprotein convertases into shorter, active forms at the Golgi. Proteases recognize specific patterns of amino acids. The cleavage often activates proteins by removing inhibitory regions.
1) Proinsulin is made as one inactive polypeptide. Convertases remove middle section, the two remaining sections form active insulin. This prevents premature signalling by insulin at the ER. This is important!
2) Regulation by proteases. ATF6 is activated by convertase proteolysis in the Golgi. The regulation is done by trafficking. BiP covers ER exit signal on ATF6 and proteases are only in the golgi.
