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Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors

Guzman, L. M.
Bost, S.
Konakova, M.
Silva, Filo
Beckwith, J.
Published in Journal of Molecular Biology. 2004, vol. 335, no. 2, p. 437-453
Abstract 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.
Keywords Adenosine Triphosphatases/metabolismAdenosine Triphosphate/metabolismAlkaline Phosphatase/metabolismAmino Acid SequenceAnimalsChickensConsensus SequenceCyclin-Dependent Kinases/genetics/metabolismEscherichia coli/metabolismEscherichia coli ProteinsFemaleGenes, Tumor SuppressorHumansMiceMolecular Sequence DataMutationOvalbumin/ physiologyPlasmidsPlasminogen Activator Inhibitor 2/ physiologyProtein Sorting Signals/ physiologyProtein Structure, TertiaryProtein TransportProteins/genetics/ metabolismProton-Motive ForceRecombinant Fusion Proteins/metabolismSequence Homology, Amino AcidSerpins/genetics/ metabolism
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PMID: 14672654
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