Membrane targeting of a folded and cofactor-containing protein

Authors

  • Thomas Brüser,

    1. Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA;
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  • Takahiro Yano,

    1. Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104-6059, USA;
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  • Daniel C. Brune,

    1. Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA
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  • Fevzi Daldal

    1. Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA;
    2. Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104-6059, USA;
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  • Note: The prefixes ‘Holo’, ‘Mal’ and ‘Apo’ are used herein solely for the description of HiPIP, the cofactor content and folded state of which has been investigated in vitro. On the other hand, the prefixes ‘pre’ and ‘mat’ are used to distinguish precursor and mature proteins. For example, the precursor of the HiPIP holoprotein is termed preHoloHiPIP, whereas a HiPIP precursor of unknown folded state is termed preHiPIP.

T. Brüser, Institut für Mikrobiologie, Universität Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany. Fax: + 49 345 5527010, Tel.: + 49 345 5526360, E-mail: t.brueser@mikrobiologie.uni-halle.de

Abstract

Targeting of proteins to and translocation across the membranes is a fundamental biological process in all organisms. In bacteria, the twin arginine translocation (Tat) system can transport folded proteins. Here, we demonstrate in vivo that the high potential iron-sulfur protein (HiPIP) from Allochromatium vinosum is translocated into the periplasmic space by the Tat system of Escherichia coli. In vitro, reconstituted HiPIP precursor (preHoloHiPIP) was targeted to inverted membrane vesicles from E. coli by a process requiring ATP when the Tat substrate was properly folded. During membrane targeting, the protein retained its cofactor, indicating that it was targeted in a folded state. Membrane targeting did not require a twin arginine motif and known Tat system components. On the basis of these findings, we propose that a pathway exists for the insertion of folded cofactor-containing proteins such as HiPIP into the bacterial cytoplasmic membrane.

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