Abstract
Cells are the entities of life and they at least consist of one aqueous compartment separated from the environment by a membrane. Lipids and proteins are important constituents of membranes and the interactions between these components are the subject of this thesis.
The studies were performed using the model organism
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Escherichia coli and membrane fractions derived thereof, as well as with pure biochemical model systems comprising phospholipids and as a model membrane protein: Leader Peptidase.
Leader Peptidase (Lep) is an integral membrane protein of E. coli and it catalyses the removal of signal peptides from translocated precursor proteins. In chapter 2 of this thesis, the number of Lep molecules per E. coli cell was determined using western blot techniques. Different strains were found to contain approximately 1000 Lep molecules per cell during exponential growth.
In this thesis (Chapter 3) it is furthermore shown that when the membrane spanning segments of Lep are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. The purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment near the catalytically region of Lep is required for the interaction with membranes.
The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. In chapter 4, orientations of constructs derived from the Lep were found to depend on the anionic (negatively charged) phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of anionic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.
In chapter 5 a cell-free system based on a lysate and membrane vesicles from E. coli is used to study characteristics of the membrane integration reaction of Lep. Integration into inverted inner membrane vesicles was detected by partial protection against externally added protease. It is concluded that integration is most efficient when coupled to translation but can also occur post-translationally and depends on the action of the proteinaceous Sec machinery and availability of anionic phospholipids. Lep is the first example of a membrane protein without cleavable signal sequence which requires anionic lipids for integration in vitro.
In chapter 6 a structural models is proposed for the action of Lep, in this model the interactions of the various domains of Lep with precursor proteins in the phospholipid bilayer, are visualised. Chapter 6 also proposes models for the interactions between anionic lipids and membrane proteins in initial stages of insertion.
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