Functional reconstitution of bacterial Tat translocation in vitro.
The Tat (twin-arginine translocation) pathway is a Sec-independent mechanism for translocating folded preproteins across or into the inner membrane of Escherichia coli. To study Tat translocation, we sought an in vitro translocation assay using purified inner membrane vesicles and in vitro synthesized substrate protein. While membrane vesicles derived from wild-type cells translocate the Sec-dependent substrate proOmpA, translocation of a Tat-dependent substrate, SufI, was not detected. We established that in vivo overexpression of SufI can saturate the Tat translocase, and that simultaneous overexpression of TatA, B and C relieves this SufI saturation. Using membrane vesicles derived from cells overexpressing TatABC, in vitro translocation of SufI was detected. Like translocation in vivo, translocation of SufI in vitro requires TatABC, an intact membrane potential and the twin-arginine targeting motif within the signal peptide of SUFI: In contrast to Sec translocase, we find that Tat translocase does not require ATP. The development of an in vitro translocation assay is a prerequisite for further biochemical investigations of the mechanism of translocation, substrate recognition and translocase structure.
Yahr
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TL
Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844, USA.
Wickner
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WT
D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015870:Gene Expression; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C408585:twin-arginine translocase complex, E coli