title: On the mode of integration of the thylakoid membrane protein cytochrome b(6) into cytoplasmic membrane of Escherichia coli.
abstract: In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present.
authors: Laboratory of Biophysics, University of Wroclaw, Wrocław, Poland.
authors: Jaroslaw
authors: J
authors: Króliczewski
authors: Beata
authors: B
authors: Gubernator
authors: Matthias
authors: M
authors: Rögner
authors: Andrzej
authors: A
authors: Szczepaniak
mesh_terms: D002462:Cell Membrane / Q000235:genetics* / Q000378:metabolism; D045303:Cytochromes b / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045347:Cytochromes b6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels; D020524:Thylakoids / Q000737:chemistry*
chemical_list: D045347:Cytochromes b6; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; D006418:Heme; D045303:Cytochromes b