Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation.
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.
Huber
Damon
D
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Boyd
Dana
D
Xia
Yu
Y
Olma
Michael H
MH
Gerstein
Mark
M
Beckwith
Jon
J
D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D057927:Hydrophobic and Hydrophilic Interactions; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D018271:Signal Recognition Particle / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D018271:Signal Recognition Particle; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases