Browsing by Subject "Sec Translokase"

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    Funktion und Dynamik eines gemeinsamen Insertionskomplexes der Sec-Translokase und YidC-Insertase in der bakteriellen Membran
    (2020) Steudle, Anja; Kuhn, Andreas
    YidC/Oxa1/Alb3-insertases and the Sec-translocase are conserved across all three kingdoms of life and constitute the most important pathway for integral proteins into cell membranes and membranes of eukaryotic organelles. The insertion of membrane proteins into the inner membrane of Gram-negative bacteria occurs mainly via the SecYEG-translocase and the YidC-insertase acting independently or in cooperation. For the cooperative insertion a close contact between SecY and YidC is assumed. Previous interaction-studies and a recently solved low-resolution structure of the so-called holo-translocon (14 Å) indicate a contact between the lateral gate of SecY and the hydrophobic substrate slide of YidC. Which specific domains of YidC and SecY thereby interact directly with each other was unknown so far. The aim of this study was to describe the contact between SecY and YidC in more detail. A high affinity for the interaction of the two proteins in detergent and in DOPC-proteoliposomes was determined via FRET measurements with fluorescently labeled SecY and YidC. For the stoichiometric ratio of the SecY/YidC-interaction a factor of one was calculated. To identify the specific contacts between SecY and YidC in vivo disulphide cross-linking experiments were performed. Direct interactions between the transmembrane domain (TM) 3 and TM8 of the SecY lateral gate and TM3 and TM5 of the hydrophobic slide of YidC were found, respectively. Furthermore, a YidC mutant with five serine substitutions, which was unable to rescue a YidC depletion strain, was investigated. Even though the serine positions are located in the middle and the periplasmic half of the hydrophobic slide of YidC and four of the positions are identical with substrate contact sites, no inhibition of insertion for the YidC-dependent substrates M13 procoat and Pf3 coat by the 5S mutant compared to the wildtype YidC was observed. For the YidC-only pathway a minimum of hydrophobicity seems to be required sufficient to allow the insertion of these substrates. In vitro FRET measurements showed an impaired interaction between SecY and the YidC 5S mutant and confirmed once again an involvement of the hydrophobic slide in the SecY/YidC-contact. Based on the cross-linking contacts and the results of the FRET measurements a possible model of the SecY/YidC-contact was established, which shows the SecY lateral gate vis-à-vis of the hydrophobic slide and the hydrophilic groove between TM3 and TM5 of YidC generating a combined SecY/YidC-cavity. Taken together, the present study provides further evidence that the lateral gate of the Sec-translocase directly interacts with the hydrophobic slide of YidC. In a further project, a SecY-YidC fusion protein was cloned to ensure the two proteins are in close proximity, the correct orientation and proper stoichiometry after reconstitution into proteoliposomes. For a collaboration with the ETH Zürich, proteoliposomes hosting the fusion protein, SecYEG, YidC or SecYEG and YidC together were prepared by myself in Hohenheim. The stepwise insertion of the Sec/YidC-dependent substrate LacY into these proteoliposomes was observed by a collaborating group of the ETH Zürich using AFM–based single-molecule force spectroscopy. The insertion of LacY was observed for the different cases but for the fusion protein and SecYEG combined with YidC the insertion process is dominated by the Sec-translocase, whereas YidC probably only has a supporting function in the folding of the protein.
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    Die Insertion des „minor coat“ Proteins G3P des Bakteriophagen M13 in die innere E. coli Membran benötigt die Insertase YidC und die Translokase SecYEG.
    (2021) Kleinbeck, Farina; Kuhn, Andreas
    The membrane of every cell forms a spacial limitation for this smallest unit of a life form. Such a very simple unicellular life form is also the Gram-negative bacterium Escherichia coli (E. coli) and is therefore a valid model organism for a living cell. Due to the inner membrane the cellular components are held together in close proximity and are separated from the extracellular environment. Most substrates cannot pass the lipid bilayer, which forms the membrane, so an import and export system had to be developed to accomplish this. For these import and export systems, very complex, polytopic transmembrane protein complexes are needed. Examples are ion channels, ion pumps or large complexes through which energy production, secretion of toxins and the transfer of nutrients are catalysed. Moreover, proteins with functions in the periplasm or outer membrane must also travel from their site of synthesis in the cytoplasm to their destination. For these different processes proteins must be inserted into or translocated across the inner membrane. Of the total proteome in prokaryotes approximately 25 to 30% is either inserted into or secreted across the inner membrane. This work identified several components required for the insertion of the "minor coat" protein G3P of M13 bacteriophage. This protein is important for the assembly of the phage particle that occurs in the inner membrane. The outermost C-terminus of G3P is anchored in the inner membrane via a single transmembrane domain, while the bulk of the approximately 42 kDa protein is located in the periplasm. Using an N-terminal cleavable signal peptide, the major portion of G3P is translocated into the periplasm via SecYEG with the help of SecA and the membrane potential. Targeting, on the other hand, could not be clearly assigned to one of the known post- or co-translational pathways. Although contact via disulfide crosslink studies to Ffh, the protein component of the ribonucleoprotein SRP, was observed via stalled ribosome nascent chains (RNCs), insertion into the membrane in vivo was independent of Ffh. Even when the interaction between SecY and FtsY, the receptor for SRP at the membrane, was impaired, G3P was inserted via SecYEG. Although the chaperone SecB was able to bind to G3P in vitro, G3P inserted independently of SecB in vivo. For membrane incorporation of G3P, it was shown that YidC is required in vivo in addition to SecYEG. Disulphide crosslink studies demonstrated that G3P first contacts the plug domain TM2b and lateral gate (TM2a and TM7) via the signal peptide of G3P, and finally the C-terminal transmembrane domain of G3P contacts YidC via TM3 and TM5 of the hydrophilic slide. Based on these contact sites, a possible insertion model was confirmed, with SecY and YidC mediating defined steps in the insertion process, providing new insights into this largely unknown process.