Browsing by Subject "Reconstitution"

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    Membraninsertion des Phagenproteins M13 procoat in Lipidvesikel mit rekonstituiertem Escherichia coli YidC
    (2011) Stiegler, Natalie; Kuhn, Andreas
    Translocation of proteins across or into the cytoplasmic membrane of Escherichia coli is accomplished by several mechanisms. The cellular secretion machinery, the translocase SecYEG, mediates the transport of unfolded proteins into the periplasm with the help of the ATPase SecA or passes the membrane proteins for bilayer integration to the insertase YidC. Membrane insertion is catalysed by YidC, whereby the native conformation of the proteins in the lipid bilayer is achieved. The translocation of a few membrane proteins occurs Sec-independently solely with the help of the insertase YidC. One of these Sec-independent proteins is the major capsid protein of the bacteriophage M13. This protein is inserted as preprotein, termed M13 procoat, with the orientation Nin-Cin into the inner membrane and a central loop domain located in the periplasm. This process is catalysed by the electrochemical membrane potential and YidC. M13 procoat is then processed by the leader peptidase to its mature form, M13 coat (orientation Nout-Cin). In the present thesis an analysis of the different transport systems of the inner membrane is performed using the example of the M13 procoat protein and its mutants. One mutant is the procoat H5EE which has 2 additional acidic residues introduced between residues +2 and +3. The insertion of this mutant requires the Sec translocase and strictly depends on the electrochemical potential. Membrane insertion of M13 procoat and derived proteins into the cytoplasmic membrane was followed in an in vitro reconstitution and translocation system. Therefore, all components of the Sec translocase (SecYEG and SecA), the insertase YidC and the different procoat proteins were purified and tested with the in vitro translocation system. Reconstitution of YidC into phospholipid vesicles depended on the lipid composition for its orientation. The cytoplasmic-out orientation corresponds to the active topology in E. coli where both termini are located in the cytoplasm. Certain lipid compositions caused the inversed orientation, which affected the catalytic activity of the reconstituted insertase. The procoat mutants H5 und H5EE were membrane inserted only in the presence of reconstituted YidC. Both proteins inserted efficiently into the vesicles with the periplasmic loop in the interior of the vesicles like the mutant PClep of procoat H5 with the C-terminal extension of the leader peptidase. Spontaneous insertion of H5 und H5EE into liposomes occurred only into leaky vesicles of the E. coli lipids. The membrane integrity was improved by the addition of an adequate amount of diacylglycerol (DAG) to the phospholipids. The leaky phospholipids were sealed by the addition of 3-4% DAG. The proteins H5 und H5EE showed a dependency of the membrane potential. Insertion occured more efficiently into YidC proteoliposomes when a stable membrane potential was generated. Proteoliposomes with reconstituted SecYEG translocase were also tested for protein insertion. Remarkedly, the protein M13 procoat H5EE efficiently inserted into SecYEG proteoliposomes, where the wildtype-like protein H5 did not.