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Bacterial phosphoenolpyruvate-dependent phosphotransferase system: association state of membrane-bound
H H Pas1, J C Ellory, G T Robillard
1Department of Physical Chemistry, University of Groningen, The Netherlands.
Abstract:
The quaternary structure of the membrane-bound mannitol permease (EIIMtl) of the bacterial phosphotransferase system in Escherichia coli has been investigated in the membrane by using the radiation inactivation method. The experiments reveal two distinct but interconvertible forms of the permease. The first state is a dimer, and the second state consists of a less active higher molecular weight complex involving the dimer. The equilibrium between these two forms in the membrane can be shifted by changing the pH. At pH 8.1 the dimer is the dominant form. Decreasing the pH results in increased binding of a regulatory protein to the dimer, thus increasing the amount of the higher molecular weight form involving the dimer. Cross-linking EIIMtl in situ, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, resulted in the formation of two cross-linked forms. One is the dimer, and the other has a higher molecular weight. Two-dimensional electrophoresis using a reversible cross-linker revealed no other protein except EIIMtl in these complexes.
Insights
The bacterial mannitol permease (EIIMtl) exists in two forms: a dimer and a higher molecular weight complex. pH influences the equilibrium between these forms, affecting EIIMtl activity in Escherichia coli.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- The bacterial phosphotransferase system (PTS) is crucial for nutrient uptake and regulation in bacteria.
- Mannitol permease (EIIMtl) is a key component of the PTS, responsible for mannitol transport.
- Understanding the quaternary structure of EIIMtl is essential for elucidating its mechanism of action.
Purpose of the Study:
- To investigate the quaternary structure of membrane-bound mannitol permease (EIIMtl) from Escherichia coli.
- To identify different functional states of EIIMtl in its native membrane environment.
- To determine the factors influencing the interconversion between these states.
Main Methods:
- Radiation inactivation method to assess molecular weight and quaternary structure in situ.
- Chemical cross-linking of EIIMtl in the membrane.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
- Two-dimensional electrophoresis with reversible cross-linkers.
Main Results:
- Two distinct, interconvertible forms of EIIMtl were identified: a dimer and a higher molecular weight complex.
- The equilibrium between these forms is pH-dependent, with the dimer favored at pH 8.1.
- Lowering the pH promotes the formation of the higher molecular weight complex through regulatory protein binding.
- Cross-linking experiments confirmed the presence of dimeric and higher molecular weight forms, with EIIMtl being the sole component.
Conclusions:
- Membrane-bound EIIMtl exists in at least two quaternary states, a dimer and a higher molecular weight complex.
- pH-sensitive interactions, involving regulatory proteins, modulate the quaternary structure and potentially the activity of EIIMtl.
- These findings provide insights into the dynamic structural organization of EIIMtl within the bacterial membrane.