Unit 4 Flashcards
what experimental technique can we use to learn about protein trafficking via vesicular transport
immunogold??
what kind of coat uses the triskelion complex
Clathrin
what does PIP do?
PIP is a regulator protein. It serves as a different signal based on its phosphorylated state
where are the following used?:
-COPII
-COPI
-Clathrin
COPII - leaving ER
COPI - leaving golgi
Clathrin - leaving PM or Golgi
why do vesicles have coats?
Helps them to assemble membrane domains and bud off
What does GTPase do in terms of coat formation?
Helps to form the COP coats and endosomal clathrin coats
are transport vesicles always spherical?
NO! Ex: collagen is too long to fit in a sphere so it has a rod-shaped vesicle
how do vesicles find their targets?
Rabs and SNARES!!
why do we use Rab-GTP fusion?
This activates and deactivates the Rabs
how do vesicles fuse to their target?
SNAREs interlock which brings them close enough to start exchanging membranes. You can only do this if both membranes have the right SNARES and Rabs
How do we return ER escapees?
They have a KDEL signal on them which tells the KDEL receptor to pick them up. This receptor attatches to a COPI vesicle and gets back to the ER where its affinity for KDEL is a lot smaller and thus it releases it before travelling back to the golgi?
What are the golgi complex regions and how do we distinguish them?
Cis Golgi Network
Cis cisternae
Medial Cisternae
Trans Cisternae
Trans Golgi Network
They are distinguished by the things they do
What activities happen in which parts of the Golgi
Cis Golgi Network
Cis cisternae
Medial Cisternae
Trans Cisternae
Trans Golgi Network
Cis Golgi Network: Sorting
+ phosphorylation of oligosacs on lysomal proteins
Cis cisternae: removes mannose
Medial Cisternae: Removes mannose, adds GluNac
Trans Cisternae: add Gal, add NANA
Trans Golgi Network: sulfates tyrosine and carbs +sorting
what is o-linked glycosylation? (include the stuff from unit 3 too)
When we add sugars onto the OHs of Ser and Thr
why do we glycosylate proteins
Prevents aggregation during folding
Protein folding timer
Protects against other macromolecules like proteases
Cell-Cell recognition:
-specificity for cell signaling
-cell-cell adhesion via lectins
What are the 2 golgi models
1) Stable Golgi
-the vesicles move through the golgi
2) Dynamic Golgi
-The vesicles from ER form the cis side of the Golgi while the prev cis side becomes medial, the medial becomes trans and so on.
-Thus as you move from cis to trans, the golgi becomes more mature
what do cells use their membranes to do
Eat, communication, respond to environmental changes
why do we call an individual cell’s membranes a “system of membranes”
Because they are interconnected either via distance, actual connection or vesicle movement
What is the difference between endo and exo cytosis
Endo: Sends things to endosome
Exo: Sends things to PM or exterior
What are the two endings an endosome can have?
Degraded by lysosome
Recycled into PM
what is an endosome?
A blob of many vesicles
do vesicles carry soluble or membrane proteins
BOTH
are vesicles selective about the type of cargo they carry and their target destinations?
Yes. VERY.
How many distinct membrane compartments use vesicular transport? Can you give some examples?
10
-gogli
-Er
-PM
what creates specificity on target compartments?
Unique combination of markers
How do target membranes create their unique combination of membrane markers?
Vesicle budding and fusion enriches or depletes the membrane of specific markers
Why must a coat be shed before fusion can occur?
So the two membranes can have direct contact
What is the difference between the inner and outer layers of a vesicle coat?
Inner layer associates the domains and attrats the second layer which helps to start the budding process
The second layer destabilizes these domains and helps to form the vesicle shape so it can be ready to bud off
If vesicles have the same coat composition, can we assume that their shape and size will be similar too?
Yes
what does COP stand for?
Coat Protein Complex
How does vesicle budding differ in the Golgi vs the PM (plasma membrane)
The plasma membrane buds are more stiff while the golgi ones are more round
What is the main component of clathrin coats?
Clathrin
what does the subunit of a clathrin coat look like?
Triskelion.
3 heavy chains with 3 light chains in between them
Can we make clathrin coats in vitro?
Yes but they are too small to hold a vesicle when we do this
What are coated-pits and on which side are they found?
They are baskets made from coat assembly. This is where the budding will start. They are found on the cystosolic side.
How are membrane proteins targeted for vesicle transport vs soluable proteins?
Membrane proteins have a cytosolic domain that interacts with the coat
soluble proteins associate with a membrane receptor that then goes on to associate with the coat
how do adaptins help with specificity? Why are they considered to be cooperatively binding?
Adaptins bind the cargo receptors and membrane cargo as well as the clathrin coat and specific PIPs (they must bind PIPs to expose their cargo receptor domains). When one binds (AP2) it bends the membrane to make further AP2 binding more probable.
How do adaptins form heterotetramers (???)
They combine diff subunits
Why is AP5 important?
AP5 is an adaptin that works on signaling for endosomes
How do adaptins work as coincidence detectors?
They add an extra layer than needs to happen for budding to occur
How specific are PIPs?
Organelle speicific and sometimes specfic to particular regions of the same membrane
Explain what membrane-bending porteins are, what they do, and how they work
Membrane bending proteins have crescent shaped BAR domains which bind to lipid heads and impose their shape. sometimes they also have a-helices that wedge into the cytoplasm and induce curvature This is used bc clathrin coats don’t make enough force on their own.
What do actin filaments do to help vesicles bud off?
They grow to add tension to help pinch off and propel the vesicle
What are PIPs?
Phosphoinositides
what % of membrane is PIPs?
10%
Where can PIPs be phosphorylated?
On the 3’, 4’ and 5’ sugars on their head
Differentiate between PI and PIP
PI is the non phosphyrlated version while PIP is the phosphorylated version
How is PI/PIP conversion regualted and compartmentalized?
This is done by
PIP phosphatases
PIP kinases
PI kinases
Can vesicular transport proteins differentiate between the PIP types?
Yes!
How do vesicles “pinch off”
Proteins like dynamin get recruited to neck.
It can bind PIP (lets it stick to membrane) and Hydrolyze GTP (regualtes pinch-rate)
It brings the two non-cytosolic sides of the leaflet together to fuse them
How do clathrin vesicles uncoat?
We package coat-dissembly factors into clathrin coats
1) PIP phosphatase depletes PIP which binds the adaptins
2)Auxilin activates the ATPase of Hsp70 chaperones which use aTP to peel the coat off
(there are additional mechanism to ensure this won’t happen till the vesicle is formed)
Happens soon after forming
How do COPI coats uncoat
Curvature triggers ARF GAP to activate
Happens soon after forming
How do COPs uncoat in general?
Coat-recruitment GTPases hydrolyse Sec 1 so it pops out of the membrane
COPII only forms if it can outrun this mechanism.
this happens soon after formation but the Coat is stable enough without sar 1 to last until it reaches its target
here, kinase phosphorylates the coat and it finally dissembles
are GTP binding proteins trimeric or monomeric?
both
what are the 3 monomeric GTPase proteins we dicussed?
ARF
Sar1
Rab
How does Sar1 form a vesicle?
Once activated, it inserts an amphiphilic helix into the cytosolic side of the ER membrane.
Then it recruits sec 23 and 24 which initiate budding. and Sec 13 and 31 which form the outer shell of the coat.
Which is more stable, COPII or clathrin coats?
COPII
are COPI coats stable?
nope. They shed soon after pinching off.
Are Rabs general or specific? Can they serve as markers?
organelle-specific.Yes.
What are Rabs main jobs?
1) Recognized by tethering proteins on target membrane
2) interact with motor proteins (Rab effectors) to move vesicle along actin filaments
3) Recruit effectors that effectively bind proteins on the target ex: SNARES
What are Rabs?
monomeric GTPases that serve as markers for vesicles and their targets.
Where do Rabs cycle between? Which form of Rab is in each destination?
cytosol -inactive
membrane - active
How does Rab-GDP become soluable in the cytosol?
Rab GDI
How does Rab-GTP insert itself into the membrane?
lipid anchor
Where do you find Rab-GEFs?
membranes of vesicles and targets
What do Rab effectors do?
membrane tethering, vesicle transport, fusions.
Only after binding active rab.
What do Rabs bind to?
many things.
Proteins that interact with SNAREs, motor or tethering proteins, ect.
How many proteins can Rab bind at once?
many
When Rabs and Rab effectors assemble onto membranes does it create a positive or negative feedback loop?
positive
What is the main job of Rabs
controlling incoming vesicular transport
What are examples of Rab effectors
SNAREs, enzymes that make or mod specific PIPs
How can the ID of an organelle be changed? Provide an example.
Ordered recruitment of sequentially acting Rabs
Rab A recruits and activates Rab B whose effectors inactivate Rab A and dissemble its membrane patch.
ex: Irreversible change from early to late endosome
Give an example of how Rabs and PIPs can work together to create an identity
Rab GEF activates Rab
Rab activates PI kinase
PI converted to PIP
PIP and active Rab bind Rab effector
Membrane patch created specialized for catching specific vesicles.
What role do Rabs play in vesicle fusion with target?
bring them close together
what happens soon after vesicular tubular clusters form?
They bud off into vesicles
what happens to clusters as they move towards the golgi?
They mature
What is retrograde transport?
escapees back to ER
How do we know what proteins need to return to the ER? (different for soluable and membrane proteins!)
soluble: KDEL
membrane: other signal on C terminus
How does the KDEL receptor pick up and drop off soluable ER resident proteins?
it has a different affinity. we don’t know how.
What mechanism keeps ER residents in the ER?
aggregation/association with other ER residents that they work with/near. This makes them too big to be transported out in a vesicle.
What will happen to an ER resident protein whose KDEL signal has been removed? (via gene editing or something)
The protein will leave the ER (but slower than a non-resident due to ER retention mechanisms)
Can proteins without KDEL signals return to the ER?
Yes some can but this return is slower than when they do have KDEL
What is a KDEL receptor?
multi-cross transmembrane protein that binds COPI coats
How do we ensure that most Golgi enzymes stay in the Golgi?
brought back faster than leaving
Why was Golgi one of the first organelles to be described?
large and regular shape
What happens if you disrupt the microtubules of a cell?
It’s golgi stacks will disperse
Does the cis side of the golgi only have cis associated proteins?
No it is ENRICHED in cis associated proteins!
What are the 2 main purposes of the golgic
sorting and processing
how do we know that the golgi is compartmentalized
different enzymes in different parts for N-linked sugar processing
What is Golgi a major site for?
carb synthesis and protein/lipid glycosylation
What are golgins
Long, filamentous tethers on the cytoplasmic side of the golgi that help to bring in vesicles by binding to their Rabs.
How are cisternae/stacks connected together?
Adjacent cisternae/stacks are connected by a scaffold of microtubule cytoskeleton proteins and cytoplasmic golgi matrix proteins (golgins?)
What is a cisterna? Can a golgi have multiple stacks? Are these stacks connected?
a flattened compartement.
Yes
Depends on the cell
do golgi enzymes work as markers?
YES!
How is the golgi taken down/rebuilt during mitosis?
matrix proteins phosphorylated so it falls apart and disperses. Then, phosphatase reverses this.
what enzymes are involved in the 5 golgi compartments?
???
What does salicylic acid do?
adds negative charge
Are most golgi proteins transmembrane or soluable?
transmembrane
What kind of proteins are the golgi’s glycosidases and glycosyl transferases?
Single-pass transmembrane proteins in multi-enzyme complexes
where do enzymatic rxns happen in the golgi?
Inner membrane
What are the 2 types of N-linked glycosylation in mammals? What determines which type will happen?
OG is trimmed and sugar is added (Complex)
OG is trimmed and nothing else (High mannose)
Determined by the location of the sugar. More mods only happen if the sugar is accesible. One protein CAN have multiple types.
What is O-linked glycosylation?
when OH groups on Ser and Thr side chains get a sugar. Usually added one at a time and typically starts with N-acetylGALATOsamine
How is mucus made?
mucins heavily glycosylated with O-linked N-GalNac
Where are Gal and Sialic acid added
Trans Golgi network and trans cisternae
What is sulfation?
when a sulfate is added giving the Tyr residue/sugar a negative charge. O-linked sugars tend to be heavily sulfated shortly after being made. ???
What do we need for sulfation?
3’phosphadenosine-5’phosphsulfate which donates sulfate. This enzyme enters trans golgi lumen from the cytosol
What are proteoglycans
a part of the ECM. Made of 1 or more glycosaminoglycan chains
How does sugar help prevent aggregation
It makes protein intermediates more soluable
How can sugar protect a protein?
Chains that are bulky and inflexible or even negative and help keep other macromolecules away and (on the PM) protect from extracellular proteases
What kind of protein helps with cell-cell adhesions and how
Lectins. They bind sugar
How can sugar help with cell/cell signaling
They can affect receptor specificity
Where do ER proteins destined for the biosynthetic-secretory pathway go?
The golgi
Where are the ER exit sites? What do they look like?
On the sER. which is distributed throughout the ER
Can improperly folded proteins leave the ER?
NO
Why do cells make more proteins than they need?
most will fail
What can be formed after vesicles leave the ER? what is required for this to happen?
vesicular tubular cluster
Why is homotypic fusion?
When vesicles derived from the same compartment fuse
Give an example of proteins that may have ER exit signals
Transmembrane proteins may have them on their cytoplasmic tail which interacts with COPII coats
Why are ER exit signals not necessary?
Everything leaves slowly without a signal. Signals do exist though to allow diff cargo to leave at diff rates and with diff efficiencies.
How do SNARES add another layer of specificity to fusion
SNAREs have to match for fusion to occur
what are SNAREs main job in fusion?
they catalyze (promote) fusion by freeing up energy
What 3 things are required for fusion to occur?
energy barrier of excluding h20 overcome
No h20
mebranes close enough together (1.5 nm)
Are SNARES specific to organells?
YES
Explain the difference between v-SNARES and t-SNAREs
v on vesicle, one chain
t on target, multiple (2-3) chains
Explain the trans-SNARE complex
the 4 chains together. It is energectically favoured and thus frees energy which can be used to drive H2O out and pull the membranes together
What are the steps of membrane fusion?
1) Lipids flow between membranes
2)Connecting stalk formed
3)lipids on other leaflets contact to amke new bilayer + fusion zone widens
4) new bilayers rupture to complete the fusion
Why would we want to delay fusion? How would we do this?
regulation.
The last part of the SNAREs is only zipped up after a specific E.C signal triggers secretion (of Ca2+??)
Does fusion still happen if a vesicle does not have the right Rab and SNAREs?
NO!
What are some examples of biological fusion?
Sperm and egg
Neurotransmitter release
Why must the SNARE complex be diassembled and reactivated B4 reuse?
The zippering of the SNAREs frees the energy needed for fusion.
What do we need to disassemble and reactivate the SNARE complex?
ATP, NSF protein and some NSF accessory proteins.
How do viruses do fusion?
they have viral fusion proteins instead of SNAREs. Once activated by the right environment, These proteins insert a partially hydrophobic patch into the host membrane
They then undergo compaction to bring the virus and host membranes closer together (like SNAREs) and drive fusion in a similar rxn to the SNAREs
How do cells speed up v/t fusion?
Extra factors help to precisly align the SNARE pairs to help initiate zippering.
