Week 11 Hitchcock Lecture 6

Question 1

What are the systems used to translocate proteins across the cytoplasmic membrane?

Answer 1

Sec and Tat

Question 2

What is the difference between the Tat and Sec system?

Answer 2

Sec translocates the protein in its unfolded state while Tat translocates proteins in their folded state.

Question 3

How does the bacterial Sec system work?

What is Sec signal sequence?

Answer 3

Preprotein is released form the ribosome - maintained in an unfolded state by the chaperone SecB. Nascent protein is bound by SecA, which recognises the N-terminal signal peptide. SecA is an ATPase and drives the transport process, ‘pushing’ the protein through the SecYEG channel. The membrane potential/proton motive force may also contribute to translocation, possible via accessory SecDFYajC complex. The N-terminal signal peptide is cleaved by a periplasmic signal peptidase

Question 4

What does Tat stand for?
What is the Tat signal sequence?
What is Tat’s composition?
How does Tat work?

Answer 4

The twin arginine translocase (Tat)

• Similar tripartite structure as Sec signal sequence but longer (26-60 amino acids, average ~30 – due to longer n- and h-regions). Canonical twin-arginine motif S/T-R-R-x-F-L-K (where x is a polar amino acid) at the n/h-region boundary. Longer and less hydrophobic h-region and basic residues in c-domain may help with ‘Sec avoidance’
• The E. coli Tat machinery comprises three proteins: TatA, TatB and TatC
• Function;
• -TatB and TatC form a complex that binds Tat substrate proteins through their signal peptides
• -TatA is recruited to the TatB-TatC complex and mediates transport of the substrate
• -TatA exists as monomers and oligomerises during Tat-mediated substrate transport
• -TatA oligomerisation may form an aqueous channel through which substrate is transported or deform/weaken membrane
• -Proteins up to ~150 kDa can be transported

Question 5

How is cytochrome c transported?

Answer 5

Some parts of the molecule are transported via Sec and some by a transporter. The apo-cytochrome is fed through the membrane by the Sec-system. Heme is exported separately from the cytoplasm by Tat system. Heme is covalently attached to the cytochrome via 2 cysteines (CXXCH) by specialised cyt c maturation system on the periplasmic side of the membrane