Browsing by Person "Klenner, Christian Daniel"

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The bacterial membrane insertase YidC : in vivo studies of substrate binding and membrane insertion
(2015) Klenner, Christian Daniel; Kuhn, Andreas
YidC of Escherichia coli belongs to the evolutionarily conserved proteins of the Oxa1/YidC/Alb3 insertase family. The transmembrane regions of the core domain, comprising of TM2-6, are the most conserved parts among the homologs and are crucial for the function as a membrane insertase. This is particularly true for the TM2, TM3 and TM5 (KUHN et al., 2003; KIEFER & KUHN, 2007). In bacteria, YidC acts as an independently working membrane insertase and, as well, in cooperation with the Sec translocon for the biogenesis of various membrane proteins. YidC is required for the biogenesis of respiratory complexes, ATP synthase and for example the mechanosensitive channel protein MscL. Also, the coat proteins of filamentous phage Pf3 and M13 require YidC for membrane insertion. The best studied substrate is the Pf3 coat protein of phage Pf3 infecting Pseudomonas aeruginosa – i.e. a small protein of 44 amino acids in length. In the context of this thesis, the YidC-dependent biogenesis of Pf3 coat was analyzed to gain better insight into the entire insertion process. In doing so, a set of more than 100 single cysteine mutants in distinct domains of YidC and Pf3 coat were generated. To study the insertion of Pf3 coat under physiological conditions, an in vivo cross-linking assay was established for capturing YidC-Pf3 interactions within a short period of time after the onset of synthesis (1 minute) using 35S-Met pulse-labelling methods. YidC binds inserting Pf3 coat protein in distinct regions of the highly conserved TM domains involving four of the six TM helices. It was verified that TM3 is indispensable for the function of YidC since four contacting residues were found in this TM helix. A helical wheel projection of substrate binding helices reveals the localization of the contacting residues of each TM segment on one helical face. This implies a helix arrangement of the transmembrane core domain which enables binding of inserting substrate proteins and interactions with transmembrane domains over the entire membrane-spanning part of YidC. The serial mutation of nine from twelve contacting residues, which are strongly hydrophobic in most cases, to serines impaired the function of YidC, whereas the single mutations had no effect. Additionally, the insertion process of translocation deficient Pf3 coat mutants was analyzed for intermediate states of the insertion process. It has been shown that the insertion deficient Pf3 coat mutants are inhibited at a late step of membrane insertion, i.e. forming the YidC contacts in the periplasmic leaflet. Based on this work, further studies confirmed that the identified substrate contacting regions of YidC play a key role in YidC-mediated insertion. The mechanosensitive channel protein MscL, M13 procoat, nascent Foc and the polytopic membrane protein LacY contact YidC at exactly the same positions (NEUGEBAUER et al., 2012; SPANN & KUHN, unpublished results; WICKLES et al., 2014; ZHU et al., 2013b).