Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors.
It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2). The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design. We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli. In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E.coli. Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized. Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the "hydrophobic core" of signal sequences. High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation. In unc- cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force. Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery.
Belin
D
D
Département de Pathologie, Université de Genève, CH-1211 Geneva, Switzerland. dominique.belin@medecine.unige.ch
Guzman
L-M
LM
Bost
S
S
Konakova
M
M
Silva
F
F
Beckwith
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D016384:Consensus Sequence; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010047:Ovalbumin / Q000502:physiology*; D010957:Plasmids; D017396:Plasminogen Activator Inhibitor 2 / Q000502:physiology*; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018892:Proton-Motive Force; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D017396:Plasminogen Activator Inhibitor 2; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; C085344:SERPIN-B5; C096854:SERPINB10 protein, human; D015843:Serpins; D000255:Adenosine Triphosphate; D010047:Ovalbumin; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D000251:Adenosine Triphosphatases