Lecture 4: Vesicles Flashcards
Learning Objectives
- What are vesicles?
- What are they used for in the cell?
- How are coated vesicles formed?
- How do cells ensure that vesicles reach the correct destination?
What are the functions of vesicles?
- communication
- respond to changes in environment
- transport (e.g. to and from Golgi)
- digestion (vesicles adapt to digest cells in immunology as a way to overcome starvation)
- storage
How are vesicles selective?
Vesicles have specific membrane proteins which signal where the vesicles needs to go. The destination has specific receptors. The cargo must also be specific.
Vesicles also have protein coats which are specific to where they are going.
What are the three types of vesicle coat?
- Clathrin: plasma membrane to Golgi and between endosomes/lysosomes and Golgi
- COPI: Golgi to ER or plasma membrane
- COPII: ER to Golgi
Discuss the structure of clathrin and clathrin coated vesicles.
Clathrin subunits are triskelion: they are composed of three large and three shape subunits. They assemble into a basket spontaneously. This drives the formation of coated pits on the cytosolic side of membrane as the basket-like lattice deforms the membrane patch, shaping the vesicle.
The clathrin molecules bind to adaptor proteins in the membrane. These adaptor proteins also have binding sites for the cargo molecule on the inside of the organelle.
Give two functions of vesicle coats.
- Concentrates specific membrane proteins in a specialised patch, which gives rise to the vesicle membrane (selecting appropriate molecules for transport)
- molds the forming vesicle - coat proteins assemble into a curved, basket-like lattice. Vesicles with the same type of coat usually have a uniform size and shape.
How do clathrin coated vesicles form?
1) The coat proteins assemble on surface of membrane. The location of where this coat starts to form on the membrane is determined by coat-recruitment GTPases (Arf1 in clathrin, Arf1 is activated when Arf1-GEF exchange the GDP on Arf1 to GTP. Arf1 then recruits protein subunits to initiate budding)
2) Protein cargo collects at membrane, bind to transmembrane receptors at lumenal side. The adaptor proteins and so clathrin are bound to the cargo receptors on the cytosolic side. Once a threshold of enough cargo has been reached, the vesicle begins to form.
3) The membrane distorts and vesicle begins to bud off
4) Clathrin coat is rapidly lost once vesicle has formed. The identity of the vesicle is now given by the transmembrane receptors.
What does dynamin do?
Dynamin is a cytoplasmic protein which regulates the pinching off of the membrane to form vesicles.
Pinching off:
- dynamin bends the patch of membrane by recruiting lipid-modifying enzymes to change the lipid composition
- a ring assembles around the neck of the bud (made of dynamin and other proteins that it recruits)
How are vesicles uncoated?
Vesicles are uncoated by PIP phosphatase (phosphatidylinositol), which weakens the binding of clathrin to the adaptor molecules. This causes the basket to disintegrate
Describe COPII coated vesicles and their formation.
COPII coated vesicles transport material form ER to Golgi.
GTP-binding proteins help mediate vesicular transport by acting as molecular switches (active = GTP bound, inactive = GDP bound)
The coat recognition GTPase Sar1 protein:
- Sar1-GEF is embedded in the ER membrane and binds to cytosolic Sar1, causing Sar1 to exchange GDP for GTP. This causes Sar1 to insert its amphiphilic helix into the cytoplasmic side of the ER membrane, tightly binding it to the membrane.
- Sar1 recruits protein subunits to the ER membrane to initiate budding
- Sar1-GTp drives COPII polymerisation (two or more COPII proteins form outer shell of coat)
GTP is important to form COPII coat - more energy is required than for clathrins.
Once the vesicle has been formed, uncoating:
- Sar1-GTP is hydrolysed to Sar1-GDP, which causes a conformational change in Sar1, which causes it to remove its amphiphilic helix from the cytosolic side of the membrane. This causes the coat to disassemble and the membrane receptors in the vesicle cell membrane are exposed and target the naked vesicle for the Golgi.
Describe COPI coated vesicles and their formation.
The COPI coat has a similar structure to clathrin.
COPI vesicles going from the Golgi to the ER or to the plasma membrane.
The coat recruitment GTPase Arf1 initiates coating once it is activated by Arf1-GEF and recruits COPI coat.
Uncoating is caused by Arf1 hydrolysis (from GTP to GDP bound state).
Give some components of the vesicle cell membrane which aid in their function.
- Adaptor proteins to bind cargo and clathrin coat proteins
- Saf1-GEF (COPII coated vesicles) and Arf1-GEF (clathrin and COPI coated vesicles)
- SNARE proteins, which allow fusion with membranes (lipid bilayers)
- vATPase, which is used to pump H+ into the vesicle, creating an electrochemical gradient.
- phopsholipids and cholesterol make up the lipid bilyare
What are Rabs and what do they do?
GTPases on the vesicle surface and target which direct the vesicle to a specific patch of the correct target membrane.
There is a particular Rab associated with each organelle, so they act as molecular markers for membrane type, e.g. Rab2 is associated with the cis Golgi network.
Rab effectors extend from the target membrane. Rab-GDP is inactive and cytosolic (it is bound to another protein called Rab-GDP dissociation inhibitor, GDI). Membrane bound Rab-GEFs exchange the GDP on Rab for GTP, activating Rab and causing it to bind tightly to the membrane. This occurs on the vesicle and the target organelle membrane.
Active Rab-GTP can bind to Rab effectors, which protrude from the target organelle membrane. This is called docking and brings the vesicle closer to the target membrane. This close proximity of the vesicle and target membrane causes the vSNARES (on vesicle) and tSNAREs (on target membrane) to become tangled and wrap around each other. The membranes become so close that they flow into each other and the closeness squeezes out any water molecules between membranes.
The SNAREs are specific to ensure only the correct membranes fuse. If the vesicle is in the wrong location, the t and v SNARES won’t interact.
Membrane fission doesn’t always happen immediately after SNAREs become tangled, e.g. in regulated exocytosis, fission is delayed until secretion is triggered by an extracellular signal.
How are SNAREs untangled?
An ATPase called NSF used the energy from ATP hydrolysis to disassemble the untangled SNAREs. Rab effectors may help in the timing and location.
How does the ER retrieval pathway work?
There are KDEL receptors in vesicular tubular clusters and the Golgi which bind to the KDEL sequence in ER proteins which have mistakenly reached the Golgi. The Er proteins are then carried to COPI coated vesicles to be transported back to the ER where they belong.
