Browsing by Person "Neugebauer, Stella"

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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.