Browsing by Subject "SecYEG"

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Membraneinbau von MscL und MscL-Mutanten aus Escherichia coli
(2012) Neugebauer, Stella; Kuhn, Andreas
About one third of all synthesized proteins in a cell are membrane proteins. To accomplish their function, it is important to ensure, that they safely reach their destination, insert efficiently into the membrane, where they fold into their correct tertiary structure. Previous studies have shown that various molecules are responsible for the targeting and insertion of membrane proteins in Escherichia coli that operate as individual modules. The mechanosensitive channel MscL is a pentameric complex in the cytoplasmic membrane of E. coli. By its action as a safety valve, MscL allows the adaption to hypoosmotic conditions of bacteria living under varying circumstances. The two transmembrane segments of the MscL monomer are connected by a periplasmic loop of 29 amino acid residues. In previous studies, the membrane insertion of MscL was analyzed in vivo in depletion strains and was monitored by modification of a single cysteine residue in the periplasmic domain of the MscL protein (Facey et al., 2007). The targeting of MscL to the inner membrane occurs in a cotranslational manner via the signal recognition particle (SRP). At the membrane, the MscL protein inserts independently of the membrane potential and the Sec-components SecAYEG, but requires YidC for the insertion process. The present thesis is about the molecular mechanisms regarding the decision whether the nascent polypeptide chain of MscL is recognized and bound by YidC or by the Sec-translocase. The periplasmic localized loop of MscL was altered by introducing negatively or positively charged residues as well as uncharged side chains and the effects on the translocation were investigated. Translocation of the periplasmic domain of MscL was detected using AMS-derivatization (4-acetamido-4´-maleimidylstilbene-2, 2´-disulfonic acid) of a single cysteine residue. The extension of the loop region by one, two or three negatively charged residues (aspartic acid residues) made the insertion of MscL dependent on the membrane potential and the Sec translocon. The requirement of SecYE was gradually affected by increasing the number of charged residues. Efficient translocation of the periplasmic loop with three additional uncharged (asparagines) residues also required the Sec-complex. The insertion of these MscL mutants was independent on the SecA component, but all the investigated mutants still showed a strict dependence on YidC. The ability of the altered MscL proteins to form functional pentameric channels was verified by growth tests and native gel electrophoresis. The presence of three additional positively charged arginine residues in the periplasmic domain inhibited MscL insertion into the lipid bilayer as well as the mutant with five additional negatively charged aspartic acid residues. As a logical consequence, the expression of these two MscL proteins could not protect the cells from osmolysis within growth tests. The direct involvement of the membrane insertase YidC with MscL and the MscL mutants was corroborated with in vivo crosslinking. YidC interacts with both transmembrane regions of MscL. Earlier studies have shown that YidC makes contact with the Pf3 coat protein in the center of the membrane. Here, the same interaction sites of YidC were identified contacting MscL during its insertion. Besides considering the significance of YidC for efficient membrane insertion, the present work has demonstrated that YidC is also essential for oligomerization of MscL into a functional channel.
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.