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A more recent version of this article appeared on March 1, 2004
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Submitted on August 18, 2003
Revised on October 28, 2003
Accepted on November 18, 2003
1 Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland, Harvard Medical School, Dept. of Cell Biology, 240 Longwood Avenue, Boston, MA 02115, U.S.A
2 Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
* Corresponding author. E-mail address: Martin.Spiess{at}unibas.ch.
Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, since their orientation in the translocon determines whether their N- or C-terminal sequence is translocated., Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the "positive-inside rule". We tested the role of conserved charged residues of Sec61p, the major component of the translocon in S.cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis using diagnostic model proteins. Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences. These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal. For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides. Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.
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