Journal article
Functional reconstitution of bacterial Tat translocation in vitro
The EMBO journal, Vol.20(10), pp.2472-2479
05/15/2001
DOI: 10.1093/emboj/20.10.2472
PMCID: PMC125449
PMID: 11350936
Abstract
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.
Details
- Title: Subtitle
- Functional reconstitution of bacterial Tat translocation in vitro
- Creators
- Timothy L Yahr - Corresponding authorwebsite: www.dartmouth.edu/∼wickner/William T Wickner - Corresponding authorwebsite: www.dartmouth.edu/∼wickner/
- Resource Type
- Journal article
- Publication Details
- The EMBO journal, Vol.20(10), pp.2472-2479
- Publisher
- Oxford University Press; Oxford, UK
- DOI
- 10.1093/emboj/20.10.2472
- PMID
- 11350936
- PMCID
- PMC125449
- ISSN
- 0261-4189
- eISSN
- 1460-2075
- Language
- English
- Date published
- 05/15/2001
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984001215102771
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