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Journal of Bacteriology, May 2005, p. 2983-2991, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.2983-2991.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Thioredoxin as a Reporter To Identify a Subset of Escherichia coli Signal Sequences That Promote Signal Recognition Particle-Dependent Translocation

Damon Huber,1 Dana Boyd,1 Yu Xia,2 Michael H. Olma,3 Mark Gerstein,2 and Jon Beckwith1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520,2 Institute for Biochemistry, Free University Berlin, 14195 Berlin, Germany3

Received 19 November 2004/ Accepted 20 January 2005

We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185:5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1920. Fax: (617) 738-7664. E-mail: jbeckwith{at}hms.harvard.edu.

Journal of Bacteriology, May 2005, p. 2983-2991, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.2983-2991.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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