Browsing by Subject "G3P"

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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.