Topological studies on the twin-arginine translocase component TatC.
The twin-arginine translocation (Tat) system can translocate folded proteins across biological membranes. Among the known Tat-system components in Escherichia coli, TatC is the only protein with multiple trans-membrane domains. TatC is important for translocon interactions with Tat substrates. The knowledge of its membrane topology is therefore crucial for the understanding of substrate binding and translocon function. Recently, based on active PhoA reporter fusions to the second predicted cytoplasmic loop of TatC, a topology with four trans-membrane domains has been suggested, calling in silico predictions of six trans-membrane domains into question. Here we report studies with translational fusions of TatC to the topological marker enzymes PhoA and LacZ which provide strong evidence for a six-trans-membrane domain topology. The stop transfer capacity of the fourth trans-membrane domain was found to be strongly influenced by the succeeding cytoplasmic domain. The presence of linker sequences at PhoA-fusion sites of the cytoplasmic domain induced PhoA leakage. In the case of one tested fusion (S185-PhoA), the stop-transfer efficiency was already low due to the negative charge in the center of the fourth trans-membrane domain (E170). The results point to the importance of cytoplasmic loops for the stabilization of stop-transfer sequences and revoke evidence for only four trans-membrane domains of TatC.
Behrendt
Jana
J
Institute of Microbiology, University of Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany.
Standar
Kerstin
K
Lindenstrauss
Ute
U
Brüser
Thomas
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015183:Restriction Mapping
D017931:DNA Primers; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C461379:TatC protein, E coli