Lecture 2 - Protein Trafficking Flashcards
Polarity must be maintained, but how?
Physical barrier
* Axon initial segment
Selective trafficking
* Polarized trafficking in neurons
and also:
Selective retention
* Protein-Protein interactions
Selective degradation
Local Translation
Neurons exhibit and retain morphological and functional asymmetry!
How does the Axon Initial Segment act as a physical barrier to maintain polarity?
Forms after axon establishment
Molecular Sieve (physical “fence”)
* Prevents free diffusion of selected
membrane and cytoplasmic proteins into axons
Regulates initiation of action potentials
What experiment was used to show the role of AIS in polarity maintenance?
Small (10 kD) and large (70 kD) soluble dye was used
The small dye could pass through but not the large dye
AIS acts as a physical barrier that allows motor proteins to selectively enter
How is protein transport in neurons similar to macro-world logistics?
Proteins and organelles (e.g. mitochondria) are in constant motion
* From sites of synthesis to sites of function (synapse formation)
* For degradation (e.g. from distal cellular regions to lysosomes located near center of the cell)
* Where energy is needed (e.g. mitochondria)
Occurs along a system of intracellular highways
* Tracks (cytoskeleton)
* Motor proteins
* Cargoes
How do we know intracellular transport exists?
Axonal transport was observed in large squid axons
Driven by microtubule tracks moving in the axon
Plus end/forward/anterograde transport - ______
Minus end/backward/retrograde transport - ______
Plus end/forward/anterograde transport - kinesin (except kinesin-3)
Minus end/backward/retrograde transport - dynein
What is the structure of a kinesin motor?
Tail
- Cargo binding
Stalk
- Mediates dimerization
Head
- Motor domain, attach to Microtubule
How do we know that kinesin ‘walks’?
In vitro motility assays
Latex beads attached to kinesin were seen to be moving
Fluorescence imaging showed the two dyes moving forward of each other - step-wise alternation
What is the mechanism propelling kinesins forward?
ATP hydrolysis by head region propels kinesins forward
Dimer with two motor heads, consisting of a catalytic core and neck linkers
Heads contain tightly bound ADP
1. Move randomly driven by Brownian motion, binds tightly to microtubule
2. ADP released and ATP enters
3. Triggers neck linker to zipper onto the catalytic core, throwing the second head forward
4. The trailing head hydrolyses ATP and releases phosphate, while the neck linker unzippers from the trailing head
And continue from 2.
How does the tail domain mediate the binding of adapters and cargoes?
Binding is regulated by post-translational modifications at the tail domain
How does the tail domain mediate autoinhibition?
The tail binds to head when there is no cargo/phosphorylation
What is the life cycle of kinesin motors?
It is degraded or recycled as dyenin
How does dynein differ from kinesin?
Dynein is a minus end motor
Dynein is much larger
The structure of the dynein complex?
2 heavy chain (HC)
* Head (motor) domain
–> ATPase activity
* Linker
–> Microtubule binding and generation of movement
* Tail
–> Dimerisation and cargo interaction
2 intermediate chains (IC)
* Scaffold linking HC with LC
* Binds Dynactin
2 light intermediate chains (LIC)
* Attached to HC
* Binds adapter proteins
2 light chains (LC)
* Assembly of dynein complex and cargo interactions
How does dynein motor motion occur?
- ATP binding releases the motor from the MT
- Bending of linker and then ATP hydrolysis
- Diffusing of motor and rebinding to MT
- Linker straightens during a powerstroke to move the whole molecule forward
- ADP release
and continue from 1.
