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Originally published In Press as doi:10.1074/jbc.M002793200 on May 17, 2000
J. Biol. Chem., Vol. 275, Issue 31, 23439-23445, August 4, 2000
Determinants of Translocation and Folding of TreF, a
Trehalase of Escherichia coli*
Kerstin
Uhland,
Martin
Mondigler,
Christoph
Spiess,
Will
Prinz §, and
Michael
Ehrmann¶
From the Fakultät für Biologie, Universität
Konstanz, 78457 Konstanz, Germany and the Department of
Microbiology and Molecular Genetics, Harvard Medical School,
Boston, Massachusetts 02115
One isoform of trehalase, TreF, is present in the
cytoplasm and a second, TreA, in the periplasm. To study the questions
of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular
compartment, we fused the signal sequence of periplasmic TreA to
cytoplasmic TreF. Even though this TreF construct was exported
efficiently to the periplasm, it was not active. It was insoluble and
degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre+ revertants of periplasmic TreF. To further
characterize periplasmic TreF, we used a genetic selection to isolate
functional TreA-TreF hybrids, which were analyzed with respect to
solubility and function. These data suggested that a domain located
between residues 255 and 350 of TreF is sufficient to cause folding
problems in the periplasm. In contrast to TreF, periplasmic TreA could
fold into the active conformation in its nonnative cellular
compartment, the cytoplasm, after removal of its signal sequence.
*
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (to M. E.) and by fellowships from the
Studienstiftung des deutschen Volkes (to M. M. and C. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Cell Biology, Harvard Medical School,
Boston, MA 02115.
¶
To whom correspondence should be addressed: School of
Biosciences, Cardiff University, Museum Ave., P. O. Box 911, Cardiff CF10 3US, United Kingdom. Tel./Fax: 44-29-2087-4648; E-mail:
ehrmann@cf.ac.uk.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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