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J. Biol. Chem., Vol. 276, Issue 39, 36501-36507, September 28, 2001
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From the Eukaryotic Microbiology, Groningen Biomolecular Sciences
and Biotechnology Institute, University of Groningen,
P. O. Box 14, 9750 AA Haren, The Netherlands and ¶ Biosi-2,
Cardiff University, Museum Avenue, P. O. Box 911, Cardiff CF10
3US, United Kingdom
Most proteins essential for the biogenesis of
peroxisomes (peroxins) that are identified to date are associated with
or are integral components of the peroxisomal membrane. A prerequisite in elucidating their function is to determine their topology in the
membrane. We have developed a novel tool to analyze the topology of
peroxisomal membrane proteins in the yeast Hansenula polymorpha in vivo using the 27-kDa NIa protease subunit from the tobacco etch virus (TEVp). TEVp specifically cleaves peptides containing the
consensus sequence,
EXXYXQ
A Novel Method to Determine the Topology of Peroxisomal
Membrane Proteins in Vivo Using the Tobacco Etch Virus
Protease*
,
, and
S
(tev). We show that cytosolic TEVp and peroxisomal TEVp.SKL are
selectively active on soluble cytosolic and peroxisomal tev-containing
proteins in vivo, respectively, without affecting the
viability of the yeast cells. The tev sequence was introduced in
between the primary sequence of the peroxisomal membrane proteins Pex3p
or Pex10p and the reporter protein enhanced green fluorescent
protein (eGFP). Co-synthesis of these functional tev-GFP tagged
proteins with either cytosolic TEVp or peroxisomal TEVp.SKL revealed
that the C termini of Pex3p and Pex10p are exposed to the cytosol.
Additional applications of the TEV protease to study peroxisome
biogenesis are discussed.
*
This work was supported by a Postdoc Universitaire
Loopbaan Stimulans grant from the Netherlands Organization for
Scientific Research through the Earth and Life Science Foundation (to
K. N. F.).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: University Hospital Groningen, Groningen
University Institute for Drug Exploration (GUIDE), Div.
Gastroenterology and Hepatology, P. O. Box 30.001, 9700 RB Groningen,
The Netherlands. E-mail: k.n.faber@med.rug.nl.
§
Supported by the Netherlands Organization for Scientific Research
through the Netherlands Technology Foundation.
Supported by Biotechnology and Biological Sciences
Research Council and German Israeli Foundation.
**
To whom correspondence should be addressed: Eukaryotic
Microbiology, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, Biological Centre, Kerklaan 30, 9751 NN Haren, The Netherlands. Tel.: 31-50-363-2176; Fax:
31-50-363-2154; E-mail:
veenhuis@biol.rug.nl.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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