Golgi Flashcards
What determines the correct targeting of intra-Golgi vesicles?
Correct SNARE interactions (these are receptors of SNAP - Soluble NSF Attachment Protein)
Rabs and Rab effectors are also essential for targeting
What are Rabs?
It is a subfamily of GTP binding/hydrolysing proteins, within the Ras superfamily.
They are monomeric but require auxiliary proteins to function (specific GEFs and GAPs)
They are cytosolic and involved in vesicle trafficking but are never part of hte mechanical coats or fusion machinery.
What are Rabs?
It is a subfamily of GTP binding/hydrolysing proteins, within the Ras superfamily.
They are monomeric but require auxiliary proteins to function (specific GEFs and GAPs)
They are found in the cytosol.
How are Rab proteins similar to SNAREs?
Each active Rab protein has a characteristic distribution on the cytosolic surface of organelle membranes:
What do Rabs do?
They help in the correct targeting of vesicles in the secretory and endocytic pathways
How do Rabs work?
Each Rab needs a GEF and a set of effectors (on the target membrane), a GAP and a GDP dissociation inhibitor (GDI).
When Rab is active/GTP-bound, Rab-effectors can bind or respond to it
What do Rab-effectors do?
They may be vesicle tethering factors or motor proteins that propel vesicles along cytoskeletal filaments to their target membrane
What is vesicle tethering?
an initial “loose” interaction of vesicles via “long” cytosolic proteins that collect and restrain the correct vesicles close to the correct target membrane
What is vesicle tethering?
an initial “loose” interaction of vesicles via “long” cytosolic proteins that collect and restrain the correct vesicles close to the correct target membrane
Tethers hold vesicles ~25nm from the target
How does tethering lead to vesicular fusion?
Free t-SNAREs would need to be in the vicinity.
They would bring the membranes closer (5-10nm apart) to promote membrane fusion.
Rabs may ‘proofread’ the SNARE pairings to make sure they are correct
How are Rabs correctly localised to recruit/bind particular effectors?
It is probably the membrane localisation of the specific Rab-GEF (the protein that adds GTP to activate the Rab) that determines exactly where the Rab operates to recruit/bind its effectors
What are the two models of the Golgi apparatus?
Vesicular transport model (stable golgi - vesicles are used to transport material in both directions)
Cisternal maturation model (unstable golgi - cisternae are constantly formed de novo and move forward, with vesicles arising just to cycle components back to the ER
Who vouched for the vesicular transport model?
Jim Rothman, Yale
What is evidence in favour of the vesicular transport model?
- The stack appears to be polarised - differential localisation of glycosylation enzymes - seen via histochemical staining
- Vesicle traffic can be reconstituted in vitro
- COPI vesicles have been shown to separately carry KDEL receptors and proinsulin. The two markers were never seen in the same vesicles suggesting a different populations of retrograde and anterograde vesicles
- Vesicles at the rims of the Golgi contain mostly cargo molecules but NOT Golgi-resident enzymes
How would you argue against the evidence in favour of the vesicular transport model?
Extensive immunoEM shows that there is a considerable degree of overlap between the localisation of golgi enzymes
How does the fact that vesicle traffic can be reconstituted in vitro?
The transport assay results were interpreted to mean that cargo (VSV G protein) had been carried forward in vesicles
But, they could be explained by the retrograde movement of the missing glycosylation enzyme to meet the cargo in an earlier cisterna
Who vouched for the cisternal maturation model?
Hugh Pelham, Cambridge
What is the evidence in favour of the cisternal maturation model?
- Rigid cell wall scales secreted from algal cells are too big to fit into 70nm vesicles and are only seen in cisternae
- Other large cargo in regular mammalian cells is also observed only in cisternae e.g. casein micelles, procollagen fibrils, VLDL
- There aren’t enough unique t- and v-SNARE partners to account for vesicle fusion to each cisterna of a stack (as well as all the ER-Golgi and post-Golgi steps)
How can you argue against the evidence in favour of the cisternal maturation model?
It could just be a peculiarity in an obscure set of unicellular algae.
Algal scales could break down at the level of each cisterna with the constituent parts being moved forward in transport vesicles followed by their rebuilding into scales in the next cisterna along
Small cargo (typical globular proteins) could still travel forwards in vesicles
Also SNAREs aren’t the only recognition factors (recall Rabs and Rab effectors) so not having enough may not be a compelling argument
What drug forces the interaction of Golgi resident proteins with ER resident proteins?
Rapamycin
Why is cisternal maturation now favoured by most people?
Sengupta et al 2015
- Because upon CHX and rapamycin treatment, all Golgi proteins bind to the ER protein and accumulate in the ER very rapidly - indicates Golgi residents recycle back to the ER.
If cargo is ‘pulsed’ into the Golgi, what would we expect according to cisternal maturation, and what was observed?
there should be a lag before it exits.
But this lag is NOT observed: cargo (VSV-G) exits the Golgi straight away
so, newly-entered cargo has the same probability of exiting than cargo already present
Who described the two-phase membrane system?
Patterson 2008
How do proteins exit the Golgi?
Exit of cargo of any size from the Golgi is exponential, not linear (=conveyor belt-like) as predicted by cisternal maturation
How do we explain the exponential manner that proteins leave the Golgi in?
cargo and Golgi enzymes partition within a continuous, two-phase Golgi, containing:
a processing domain
an export domain
these domains would be defined by asymmetrical lipid composition
What does the two-phase membrane system explain?
- exponential kinetics of export
- waves of cargo observed
What does the two-phase membrane system fail to explain?
Spatial separation and sequential action of glycan-processing enzyme
How are golgi enzymes selectively retained?
- proteases have retrieval signals
- ‘kin recognition’ identifies golgi proteins and aggregates them to they cannot enter transport vesicles
- TGN glycosyltransferases have short TM spans to prevent them entering the ‘thicker’ cholesterol rich membrane regions that are budding off to cell surface
How are some proteins isolated to be transported to the cell surface?
Lipids are sorted into microdomains or ‘lipid rafts’.
These lipids are saturated and rich in cholesterol so make the bilayer appear thicker.
Certain proteins with longer TMDs can fit in these lipids
What are the two types of secretion and where do they occur?
Constitutive secretion occurs in all cells
Regulated secretion in some cell types.
How do proteins go through regulated secretion?
Cargo concentrates in the TGN around nucleation factors.
Proteins continue to mature and condense in secretory vesicles or granules
Signalling events are needed to trigger fusion of these granules with the plasma membrane as protein ‘clamps’ prevent the final interactions of granule v-SNAREs with membrane t-SNAREs.
How was transport through the Golgi investigated?
By trapping proteins that travel through the Golgi by inducing aggregates
If the protein is still able to travel through the Golgi, it supports cisternal maturation.
If the protein is stuck, it supports vesicular transport.
What was the main issue with comparing how Rothman and Luini investigated golgi transport?
They looked at the transport of different proteins.
Rothman = CD8 which travels to plasma membrane if monomeric, 4FM (FKBP mutant - aka staples)
Luini = MANI which is a resident of cis-medial Golgi membrane, 3FM
What was Rothman’s method in investigating golgi transport?
Accumulate monomeric CD8 (+AP) in cis Golgi By 16C temperature block Wash out AP, raise temperature See where it is and where it goes Re-add AP and see what happens
What was Luini’s method in investigating golgi transport?
Wash out AP and let MANI-FM polymerise
See where it is and where it goes
Re-add AP, to convert MANI-FM back into monomers and see what happens
What was the result of Rothman’s investigation into golgi transport?
After removing AP (reverses stables) and raising T, polymeric CD8 is stuck in the cis-Golgi at first - but both VSVG and collagen still traffic normally and Golgi appears normal.
Re-adding AP allowed CD8 to travel via TGN to PM.
FM-aggregates of soluble proteins can still travel through the stack by entering vesicles at the rim of the cisternae.
What was the result of Luini’s investigation into golgi transport?
Removing AP causes MANI-FM to disappear from vesicles and tubular carriers. Polymeric MANI-FM moves to the trans Golgi instead. VSVG still traffics normally and Golgi appears normal.
Re-adding AP causes MANI-FM to return to cis-medial Golgi by entering vesicles/carriers
What was the conclusion of Rothman’s investigation into golgi transport?
CD8 accumulates in the cis Goldi and cannot move but aggregates of soluble proteins can still move through the rims.
This supports vesicular transport.
What was the conclusion of Luini’s investigation into golgi transport?
MANI travels to trans-Golgi by cisternal maturation with polymeric. It then enters the retrograde vesicle flow when monomeric.
This supports cisternal maturation
Who is right? Luini or Rothman?
Luini’s is more straightforward.
Rothman pays much more attention to how staples are formed (trans-aggregates) whereas Luini postulates the formation of cis-aggregates.
Rothman’s system obviously suffers from overexpression, whereas Luini is more careful with expression levels
But Rothman has more controls!
