Lecture 7: Sec & Tat Flashcards
What 2 systems operate in parallel in bacteria that can transport a large proein without too much influx of ions?
Biggest difference?
2 systems operating in parallel in bacteria that can do this.
- Sec system (universally conserved. Unfolded proteins)
- Tat system (protein folded in cytoplasm before it is transported)
What does Signal sequence for crossing IM look like?
These signal sequences have a tripartite structure: Polar N-terminal = synthesized first. Then, hydrophobic region. Then, polar sequence (C). Cleavage = at end of polar sequence (C): AxA
Sec- pathway: features?
> Important: it has to be unfolded for the Sec-pathway to be started
Post-translational translocation (eukaryotes + bacteria)
Also involved in co-translational insertion of MP recognized by SRP. Ribosome stops making new protein until it docks onto the Sec machinery
How does translocation via Sec go?
- L23: recognizes signal peptide of the protein synthesized by the ribosome (yellow). Holds on to it
- Deformation of the ribosome structure and the protein synthesized is pushed out to form a loop.
- Leads to the recruitment of a chaperone called SecB -> recognizes unfolded protein structure and binds to it to prevent folding or interaction with other proteins.
- Sec machinery: SecA binds to SecB complex, signal peptide inserted into sec machinery into the membrane and is cleaved by leader enzyme peptidase. Accumulated in membrane
- Polypeptide pushed through the channel with each ATP being hydrolysed
What is difference in signal sequence for Tat vs Sec?
Tat = > N region longer, hydrophobic region less hydrophobic, always find 2 arginine’s at end of polar N region. (Twin Arginine Translocation)
What happens when mutating 2 argenines?
two arginines are essential for transport. Mutation of one arginine to lysine
(which is also positively charged) reduces transport efficiency by ± 50%. Mutating
both arginines completely blocks transport.
TAT characteristics?
- Transports folded proteins across an ion-tight membrane.
- No ATP is hydrolyzed (PMF).
- Substrates often contain cofactors.
- Substrates are sometimes dimers, where only one has a
- signal peptide. Yet they are only transported together.
- Diameters of substrates up to 8 nm.
- Tat is essential for virulence of many pathogens.
- Does not occur in mammals. Homolog in plant chloroplasts. Good target!
No of Tat substrates differs tremendously. Why is it used?
In general, scientists believe that proteins that are targeted to the Tat system
either require assistance from chaperones in the cytoplasm to fold, bind the
correct ligand and become active, or they fold too quickly, which renders
them incompatible with the Sec system.
Examples for proteins of why Tat system is used?
- binding of complex cofactors (must happen in the cytoplasm)
- specificity of metal insertion (metal ions as cofactors) (cytoplasm to have control over this)
- Hetero-oligomerization
Components of the Tat system? Which are interchangeable and why?
- 4 components: TatA,B,C,E. ABC in the same operon, E in another. A and E are interchangeable: TatE is a gene duplication of TatA. ABE = amphipathic (hydrophobic amino-acids inside, hydrophilic outside). TatC = largest
-> Large variation between species. Number of Tat components, number of substrates
TatB function seems to be placed on the TatA in organisms that do not have a TatB themselves.
Function of TatA complex?
Structure of TatA complex
TatA assembles into large complexes of various sizes (for different proteins). It Is therefore believed to form the pore through the membrane.
Most abundant
Structure TatA?
There is no structure of the entire complex -> might be highly variable for different proteins of different shapes and sizes.
- TatA transmembrane helix is too short
Currently most accepted model of Tat system? Polymerization model?
is no stable complex, to prevent accidental opening of a huge pore. Only when a substrate binds to TatC, TatB moves a bit, triggering recruitment of TatA monomers, which assemble into a pore. After translocation, the complex disassembles again.
What is not in agreement with current model regarding substrate-tat interaction?
Translation inhibition (no substrate-Tat interaction): reduced no of Tat-complexes, but still some Tat-complexes left, which is not in agreement with the current model.
On video, you never see assembly-disassembly, only very stable complexes TatA system diffuses. Diffusion decreases with larger radius. TatA diffuses too fast for its size. How?
- distortion of lipids to break viscosity-imposed speed limit of membrane diffusion
- short trans-membrane domain
Rhomboid protein: distorts lipids to break the viscosity-imposed speed limit of membrane diffusion. Make the membrane thinner. That’s why they can diffuse with great speed. Perhaps that happens to TatA as well. -> Rhomboids diffuse too rapidly due to their short trans-membrane domain. TatA has a too short transmembrane domain. This is essential for its function.
Relationship between short helix, rapid diffusion & function: transporting folded proteins across the membrane?
