Biogenesis of a respiratory complex essential for viability of pathogenic Mycobacteria
Lead Research Organisation:
University of Dundee
Department Name: College of Life Sciences
Abstract
Most of the organisms on earth require oxygen for life. This is not just true for animals, but also for bacteria, and it supports an essential process known as respiration. During respiration electrons (derived from food such as glucose) are used to reduce the oxygen to water. This process happens within the membranes of cells and is carried out by proteins that are found embedded within those membranes. The proteins accept the electrons that derive from glucose and pass them along a chain, at the end of that chain oxygen is reduced and water is formed.
One of the essential components of the electron transfer chain is three proteins that interact with each other and is collectively called the cytochrome bc1 complex. Each of the three proteins of this complex binds a metal cofactor that can accept the electrons and pass them on to the next protein in the chain. We are interested in one of the proteins of the cytochrome bc1 complex that is called the Rieske protein. This protein contains a special type of cofactor called an iron sulphur cluster that is essential to allow the protein to transfer electrons.
We have discovered that the Rieske protein from the pathogen Mycobacterium tuberculosis, which causes the deadly disease tuberculosis, and some other closely related bacteria is unusual, because it is much bigger than expected. This means that the Rieske protein in these bacteria has to be inserted into the membrane in a different way than the Rieske protein from all other life forms. We would like to understand how these bigger Rieske proteins are made. In addition to this we would like to find out what this extra part of the Rieske protein is doing, and whether it is essential to allow these bacteria to respire oxygen. One way we will do this is to remove this extra part and see if it stops the Rieske protein from working. Another way we will do this is to examine the organisation of the cytochrome bc1 complex when we interfere with the Rieske protein by preventing it from being properly inserted into the membrane.
Ultimately the work we will do in this project will give us fundamental insight into how this protein complex is assembled. This basic knowledge that we generate may eventually be exploited to interfere with the assembly of this essential complex in Mycobacterium tuberculosis.
One of the essential components of the electron transfer chain is three proteins that interact with each other and is collectively called the cytochrome bc1 complex. Each of the three proteins of this complex binds a metal cofactor that can accept the electrons and pass them on to the next protein in the chain. We are interested in one of the proteins of the cytochrome bc1 complex that is called the Rieske protein. This protein contains a special type of cofactor called an iron sulphur cluster that is essential to allow the protein to transfer electrons.
We have discovered that the Rieske protein from the pathogen Mycobacterium tuberculosis, which causes the deadly disease tuberculosis, and some other closely related bacteria is unusual, because it is much bigger than expected. This means that the Rieske protein in these bacteria has to be inserted into the membrane in a different way than the Rieske protein from all other life forms. We would like to understand how these bigger Rieske proteins are made. In addition to this we would like to find out what this extra part of the Rieske protein is doing, and whether it is essential to allow these bacteria to respire oxygen. One way we will do this is to remove this extra part and see if it stops the Rieske protein from working. Another way we will do this is to examine the organisation of the cytochrome bc1 complex when we interfere with the Rieske protein by preventing it from being properly inserted into the membrane.
Ultimately the work we will do in this project will give us fundamental insight into how this protein complex is assembled. This basic knowledge that we generate may eventually be exploited to interfere with the assembly of this essential complex in Mycobacterium tuberculosis.
Technical Summary
The ability to respire oxygen is essential for the growth of Mycobacterium tuberculosis. The cytochrome aa3 oxidase is the end point of the M. tuberculosis respiratory chain, and the site at which oxygen is reduced to water. The electrons that are used to reduce oxygen are provided by the quinol pool in the cytoplasmic membrane, and are fed into the aa3 oxidase via the essential quinol:cytochome c oxidoreductase (bc1 complex).
We have good evidence that one of the components of the M. tuberculosis cytochrome bc1 complex, the Rieske iron-sulphur protein, has unique features and a highly unusual mechanism of assembly into the membrane. Unlike all other Rieske proteins, which have a single transmembrane helix, the Rieske protein of M. tuberculosis and other members of the Actinobacteria has three transmembrane helices. The iron-sulphur cluster-containing domain in all bacterial Rieske proteins is transported across the membrane by the Tat pathway, which recognizes the Rieske transmembrane domain as a signal for export. Interestingly, we have shown that the Tat pathway recognizes the third transmembrane helix of the Actinobacterial Rieske protein as the signal to transport the iron-sulphur cluster-containing domain across the membrane. This means that the first two transmembrane helices are integrated into the membrane in a Tat-independent way and that two different protein transport systems participate to assemble this protein. We aim to elucidate:
a. which translocases participate in the assembly of the 3TMs of the Rieske protein and
b. How an internal twin arginine signal peptide is recognized by the Tat pathway.
It is not clear why the Rieske protein of the cytochrome bc1 complex is larger than those of all other organisms. We aim to elucidate the role of the extra hydrophobic part of the protein in the assembly and stability of the Actinobacterial cytochrome bc1 complex by completing the following four objectives:
c. Examine the requirement for TMs 1 and 2 for in vivo Rieske function
d. Probe the Localization and topology of the Rieske protein in the model Actinobacterium Streptomyces coelicolor.
e. Assess the requirement of TMs 1 and 2 for bc1 complex assembly and/or stability
f. Develop a strategy for the purification of the Actinobacterial cytochrome bc1 complex
We have good evidence that one of the components of the M. tuberculosis cytochrome bc1 complex, the Rieske iron-sulphur protein, has unique features and a highly unusual mechanism of assembly into the membrane. Unlike all other Rieske proteins, which have a single transmembrane helix, the Rieske protein of M. tuberculosis and other members of the Actinobacteria has three transmembrane helices. The iron-sulphur cluster-containing domain in all bacterial Rieske proteins is transported across the membrane by the Tat pathway, which recognizes the Rieske transmembrane domain as a signal for export. Interestingly, we have shown that the Tat pathway recognizes the third transmembrane helix of the Actinobacterial Rieske protein as the signal to transport the iron-sulphur cluster-containing domain across the membrane. This means that the first two transmembrane helices are integrated into the membrane in a Tat-independent way and that two different protein transport systems participate to assemble this protein. We aim to elucidate:
a. which translocases participate in the assembly of the 3TMs of the Rieske protein and
b. How an internal twin arginine signal peptide is recognized by the Tat pathway.
It is not clear why the Rieske protein of the cytochrome bc1 complex is larger than those of all other organisms. We aim to elucidate the role of the extra hydrophobic part of the protein in the assembly and stability of the Actinobacterial cytochrome bc1 complex by completing the following four objectives:
c. Examine the requirement for TMs 1 and 2 for in vivo Rieske function
d. Probe the Localization and topology of the Rieske protein in the model Actinobacterium Streptomyces coelicolor.
e. Assess the requirement of TMs 1 and 2 for bc1 complex assembly and/or stability
f. Develop a strategy for the purification of the Actinobacterial cytochrome bc1 complex
Organisations
People |
ORCID iD |
| Tracy Palmer (Principal Investigator) |