Vesicle Trafficking Flashcards
Why is vesicular trafficking needed?
Electron microscopy highlights cellular structures and shows that the cytosol is not empty and packed full of organelles and molecules.
What are the two types of vesicle mediated transport?
Selective retrieval by donor compartment (KDEL)
Selective retention by acceptor compartment
Explain the cell free assay biochemical approach that shows the use of vesicular trafficking in Golgi transport.
- Lack of GlcNAc transferase blocks 3rd step (medial cisternae) in golgi modifications, leaves protein with high mannose content.
- Endo H cleaves high mannose content proteins. (Sensitivity)
- Purified golgi lacking NAGT infected with VSV, doesn’t fully process viral glycoprotein.
- Adding a WT Golgi, fully processes the protein and completes modifications.
- Cell becomes Endo H resistant, shows vesicular trafficking.
Explain the yeast genetic approach to identify the components of the secretory pathway.
Temperature sensitive yeast mutant cells stop growing at 35 degrees and vesicular intermediates accumulate.
Isolate congested cells and mate to create diploid cells.
If there is a high copy number of interacting genes, cells can be rescued and continue growing.
Can see which genes caused rescue - showed GTP coating/uncoating and NEM/ATP dependent fusion.
What are the three different types of coat proteins and where do they target vesicles to and from.
Clathrin - PM endocytic vesicles to endosome, TGN to late endosome.
COPI - retrograde Golgi –> ER
COPII - anterograde ER –> Golgi
Describe the process of endocytosis.
Adaptor proteins recognise the cargo and recruit Clathrin, which forms a triskelion cage around the cargo.
Membrane fission occurs, aided by Dynamin, forms a Clathrin coated vesicle.
Vesicle uncoats by chaperone proteins - Hsc70 and Auxillin.
State the structure of Dynamin and how it works.
GTPase-Middle-PH-GED-PRD
Dynamin builds a contractile ring around the neck of the forming vesicle. GTPase activity causes conformational change for membrane scission.
Describe the process of exocytosis.
Cargo recruits a COPII coat.
Sar1pGDP binds Sec12 (GEF), causes GTP exchange to create Sar1pGTP.
This recruits Sec23-24p dimer. 24p binds cargo, 23p is GAP.
Complex recruits Sec13-31 complex as secondary coat monomer. Binding repositions Sec23p to optimise hydrolysing activity.
GTP hydrolysis occurs and coated vesicle forms, Sec13-13 polymerises.
Describe the motor proteins.
KINESIN - +end directed, anterograde. 2 heavy, 2 light chains. Light chain has TPRs to interact with cargo. 8nm ATP driven steps.
DYNEIN - -end directed, retrograde. 2large/2int/2small subunit structure. Cargo binding regulated by Dynactin. Step size dependent on load, 8nm when attached to vesicle. Dynein motors are cross-regulatory, to ensure correct directionality.
What do tethers do and what are the two types (with examples)?
Tethers bring two membrane close together to initiate docking/fusing.
LONG COILED TETHER - p115 - recognises Rab1 on vesicle to bring it to the Golgi membrane.
HETEROOLIGOMERIC COMPLEX - Exocyst - 8 subunit complex, targets post-golgi vesicles for PM fusion / exocytosis.
Sec15p (vesicle association), Exo70/84 (PM interaction)
What are the major components of the fusion machinery?
SNARES - SNAP receptors.
NSF - NEM sensitive fusion protein.
SNAP - soluble NSF attachment protein.
How were the components of SNARE proteins found and what are they?
Immobilise NSF on column
Add SNAP
Run brain homogenate through column under non-hydrolysing ATP conditions.
Elute the bound receptors by allowing ATP hydrolysis, identifies NSF-SNAP interacting proteins
Synaptobrevin, Syntaxin, SNAP-25
Describe the formation of a SNARE bundle for exocytosis at the synaptic membrane, fusion and recycling.
A SNARE bundle is always 4 helices. 3 helices provided by the target (1 from Syntaxin, 2 from SNAP-25) and 1 provided by the vesicle (Synaptobrevin).
Zipper up in a high affinity state, Trans-SNARE complex - provides energy for fusion to the Cis-SNARE complex.
Recycling - Break interactions with NSF-SNAP and ATP activity. Twist and unwind bundle.
What proteins aid SNARE bundle formation?
Sec-Munc 18-like proteins
Absolutely required for fusion. Bind syntaxin so it cannot bind SNARES.
When engaged with other SNARES,bind syntaxin N-terminus to associate a more active conformation and promote membrane fusion.
What is the role of Synaptotagmin 1?
A calcium sensor, that in the presence of calcium, removes complexin that blocks the zippering up of partial SNARE bundles.
They hold the helices in a partial ring bundle, so when activated, can easily move the full bundle into position.
